Methods of using variants of fgf19 polypeptides for the treatment of cancer

ABSTRACT

Models useful in determining whether fibroblast growth factor 19 variant polypeptides having glucose-lowering activity and/or anti-obesity activity also exhibit favorable oncology-related profiles, and methods and uses associated therewith. Also provided are methods of antagonizing the oncogenic activity of FGF19 in a subject and, in certain embodiments, methods of preventing or treating a disease, disorder or condition, such as a FGF19-dependent disease, disorder or condition, or a symptom thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/032,605, filedJul. 25, 2016, which is a U.S. National Stage Application under 35U.S.C. § 371 of International Patent Application No. PCT/US2014/062378,filed Oct. 27, 2014, which claims the benefit of U.S. Ser. No.61/896,473 filed Oct. 28, 2013, U.S. Ser. No. 61/922,586 filed Dec. 31,2013, and U.S. Ser. No. 62/067,273 filed Oct. 22, 2014, each of which isincorporated herein by reference in its entirety.

FIELD

The invention relates to, among other things, models useful indetermining whether polypeptide variants of a fibroblast growth factorhaving glucose-lowering activity also exhibit favorable oncology-relatedprofiles, and methods and uses involving the foregoing. Also providedare methods of antagonizing the oncogenic activity of FGF19 in a subjectand, in certain embodiments, methods of preventing or treating adisease, disorder or condition, such as a FGF19-dependent disease,disorder or condition, or a symptom thereof.

BACKGROUND

Diabetes mellitus is a debilitating metabolic disease caused by theabsence of insulin production (type 1), or insulin resistance orinsufficient insulin production (type 2) from pancreatic β-cells,endocrine cells that manufacture and store insulin for release followinga meal. High blood glucose levels stimulate the secretion of insulin bypancreatic β-cells. Insulin, in turn, stimulates the entry of glucoseinto muscles and adipose cells, leading to the storage of glycogen andtriglycerides and to the synthesis of proteins. Activation of insulinreceptors on various cell types diminishes circulating glucose levels byincreasing glucose uptake and utilization, and by reducing hepaticglucose output. Disruptions within this regulatory network can result indiabetes and associated pathologic conditions.

An individual having a glucose metabolism disorder can suffer fromhyperglycemia, hyperinsulinemia, and/or glucose intolerance, along witha host of related disorders. For example, insulin resistance, a disorderoften associated with aberrant levels of glucose and/or insulin, ischaracterized by hepatic, fat, and muscle cells losing their ability torespond to normal blood insulin levels. Such glucose metabolismdisorders adversely affect a large and growing number of individualsthroughout the world.

Obesity, which is most commonly caused by excessive food intake coupledwith limited energy expenditure and/or lack of physical exercise, oftenaccompanies various glucose metabolism disorders. Obesity increases thelikelihood of an individual developing various diseases, such asdiabetes mellitus, hypertension, atherosclerosis, coronary arterydisease, gout, rheumatism and arthritis. Moreover, mortality riskdirectly correlates with obesity, such that, for example, a body-massindex in excess of 40 results in an average decreased life expectancy ofmore than 10 years.

Certain pharmacological treatment modalities have demonstrated, tovarying degrees, both glucose homeostatic and anti-obesity activity.Unfortunately, such modalities are frequently associated with seriousand often debilitating adverse effects.

In view of the prevalence and severity of diabetes, obesity, andassociated metabolic and non-metabolic disorders, along with theshortcomings of current treatment options, alternative treatmentmodalities are needed.

SUMMARY

Bariatric surgery has been proposed as an alternative,non-pharmacological treatment for diabetes. It has been postulated thatchanges in gut hormone secretion following surgery are responsible forthe resolution of diabetic conditions. Serum levels of Fibroblast GrowthFactor 19 (FGF19) in humans are elevated following gastric bypasssurgery. FGF19 is highly expressed in the distal small intestine, andtransgenic over-expression of FGF19 improves glucose homeostasis(Tomlinson, E. (2002) Endocrinology 143(5):1741-47). Augmentedexpression and secretion of FGF19 could at least partially explain theremission of diabetes observed following surgery.

Despite the desirable metabolic effects attributable to FGF19 (e.g.,blood glucose lowering), treatments that increase FGF19 levels (through,for example, enhancement of FGF19 expression or administration ofexogenous FGF19) are associated with induction of hepatocellularcarcinoma (HCC). Thus, there is an on-going effort to identify agentsthat possess the favorable characteristics of FGF19 without inducingcancerous conditions like HCC. The present disclosure is based, in part,on animal models and associated methods to assist in the accurate andefficient determination of whether a candidate agent possesses suchattributes and whether a subject is a viable candidate for suchtreatment.

In further embodiments, a use or method of treatment of a subject isintended to or results in reduced glucose levels, increased insulinsensitivity, reduced insulin resistance, reduced glucagon, animprovement in glucose tolerance, or glucose metabolism or homeostasis,improved pancreatic function, or reduced triglyceride, cholesterol,intermediate density lipoproteins (IDL), low density lipoproteins (LDL)or very low density lipoproteins (VLDL) levels, or a decrease in bloodpressure, a decrease in intimal thickening of the blood vessel, or adecrease in body mass or weight gain.

In one embodiment, the present disclosure contemplates a method fordetermining whether a test subject having a metabolic disorder is acandidate for treatment with a FGF19 variant, the method comprising a)co-administering FGF19 or a FGF19 surrogate, and a FGF19 variant to thetest subject having a metabolic disorder, wherein the amount of theFGF19 or the FGF19 surrogate administered to the test subject issufficient to induce a cancerous condition in a reference population,and b) determining whether an indicia of a cancerous condition isobserved in the test subject; wherein the absence of an indicia of acancerous condition indicates that the test subject is a candidate fortreatment with a FGF19 variant.

As used herein, the term “FGF19 surrogate” is meant to include anymolecule (e.g., a polypeptide) capable of eliciting a same or acomparable effect as FGF19, wherein the effect is generallycancer-related (e.g., the induction of tumor formation or any otherindicia of a cancerous condition). An FGF19 surrogate is frequently avariant of FGF19, including active fragments, having at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 93%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, amino acid sequence identityto a contiguous stretch of from about 150 amino acids to about 160 aminoacids, from about 160 amino acids to about 170 amino acids, from about170 amino acids to about 180 amino acids, from about 180 amino acids toabout 190 amino acids, or about 194 amino acids or more, of one of theamino acid sequences described herein.

In the present disclosure, the phrase “an indicia of a cancerouscondition” broadly refers to any indication that a cancerous disease,disorder or condition has formed, is forming or is likely to form. Mostcancers are initially recognized either because of the appearance ofsigns or symptoms or through screening. Definitive diagnoses generallyrequire, among other means, one or more of pathological examination of atissue sample, blood tests, x-rays, CT scans and endoscopy. Cancerousconditions refer to any type or classification of cancer, includingcarcinomas, sarcomas, lymphomas and leukemias, and blastomas.

Cancer symptoms are usually caused by the effect of a cancer on the partof the body where it is forming (e.g., unusual lumps on the breasts orchanges in moles on the skin), although cancerous diseases, disordersand/or conditions may cause more general symptoms such as weight loss orfatigue. In the methods and models described herein, an indicia of acancerous condition (or disorder or disease) is frequently a tumor(e.g., a colon tumor or a hepatic tumor). Observations and measurementsof a reduction in tumor number, tumor size, or tumor weight frequentlyindicate that a treatment modality is having a positive effect.

In particular embodiments, the indicia of a cancerous condition is/areassociated with hepatocellular carcinoma (HCC, also referred to asmalignant hepatoma), the most common type of liver cancer. HCC maypresent with jaundice, bloating from ascites, easy bruising from bloodclotting abnormalities, loss of appetite, weight loss, abdominal pain,nausea, emesis or fatigue. HCC is discussed further hereafter.

In another embodiment, the present disclosure contemplates a method fordetermining whether a test subject having a metabolic disorder is acandidate for treatment with a FGF19 variant, the method comprising a)providing a test subject having an indicia of a cancerous condition, thesubject having a metabolic disorder, b) co-administering FGF19 or aFGF19 surrogate, and a FGF19 variant to the test subject, wherein theamount of the FGF19 or the FGF19 surrogate administered to the testsubject is sufficient to induce a cancerous condition in a referencepopulation, and c) determining whether an indicia of a cancerouscondition is enhanced in the test subject; wherein the absence ofenhancement of an indicia of a cancerous condition indicates that thetest subject is a candidate for treatment with a FGF19 variant.

In a further embodiment, the present disclosure contemplates a methodfor determining whether a test subject having a metabolic disorder is acandidate for treatment with a FGF19 variant, the method comprising a)providing a test subject having an indicia of a cancerous condition, thetest subject having a metabolic disorder, b) and co-administering FGF19or a FGF19 surrogate, and a FGF19 variant to the test subject, whereinthe amount of the FGF19 or the FGF19 surrogate is administered to thetest subject is sufficient to induce a cancerous condition in areference population, and c) determining whether an indicia of acancerous condition is reduced in the test subject; wherein thereduction of an indicia of a cancerous condition indicates that the testsubject is a candidate for treatment with a FGF19 variant.

The present disclosure also contemplates a method for determiningwhether a FGF19 variant is a candidate for treating a test subjecthaving a metabolic disorder, the method comprising co-administeringFGF19 or a FGF19 surrogate, and the FGF19 variant to the test subjecthaving a metabolic disorder, wherein the amount of the FGF19 or theFGF19 surrogate administered to the test subject is sufficient to inducea cancerous condition in a reference population, and determining whetheran indicia of a cancerous condition is observed in the test subject;wherein the absence of an indicia of a cancerous condition indicatesthat the FGF19 variant is a candidate for treatment of the test subject.

Other embodiments contemplated herein are drawn to a method fordetermining whether a FGF19 variant is a candidate for treating a testsubject having a metabolic disorder, the method comprising providing atest subject having a metabolic disorder, the test subject having anindicia of a cancerous condition, co-administering FGF19 or a FGF19surrogate, and a FGF19 variant to the test subject, wherein the amountof the FGF19 or the FGF19 surrogate is administered to the test subjectis sufficient to exacerbate a cancerous condition in a referencepopulation, and determining whether an indicia of a cancerous conditionis enhanced in the test subject; wherein the absence of exacerbation ofan indicia of a cancerous condition indicates that the FGF19 variant isa candidate for treatment of the test subject. In particularembodiments, one or more indicia of a cancerous condition are reduced inthe test subject.

In still further embodiments, the present disclosure contemplates amethod of treating (or preventing, in certain circumstances) a subjecthaving a metabolic disorder, the method comprising providing a subjecthaving a metabolic disorder, wherein the subject exhibits an indicia ofa FGF19-induced cancerous condition, and administering to the subject atherapeutically effective amount of a FGF19 variant identified from apool of candidate FGF19 variant polypeptides as described herein;wherein there is an improvement in the metabolic disorder in thesubject.

As alluded to above, the present disclosure also contemplates variousmodels. One embodiment is directed to a model for determining whether aFGF19 variant is a candidate for preventing a cancerous disease,disorder or condition in a subject having a metabolic disorder, themodel comprising a subject that i) does not exhibit an indicia of acancerous condition prior to the administration of an effective amountof a FGF19 or FGF19 surrogate, and ii) exhibits an indicia of acancerous condition after the administration of FGF19 or FGF19surrogate; and wherein an indicia of a cancerous condition improves uponadministration of an effective amount of a polypeptide comprising anamino acid sequence set forth in SEQ ID NO:1. In certain embodiments,the polypeptide consists of an amino acid sequence set forth in SEQ IDNO:1.

The present disclosure also contemplates a model for determining whethera FGF19 variant is a candidate for treating a cancerous disease,disorder or condition in a subject having a metabolic disorder, themodel comprising a subject having at least one indicia of cancerresulting from administration of FGF19 or FGF19 surrogate, wherein theindicia of cancer improves upon administration of an effective amount ofa polypeptide comprising an amino acid sequence set forth in SEQ IDNO:1. In certain embodiments, the polypeptide consists of an amino acidsequence set forth in SEQ ID NO:1.

Though not limiting, in certain embodiments, the FGF19 variant is M70(SEQ ID NO:1) in the methods and models of the present disclosure. AnFGF19 variant can be identified from a pool of candidate FGF19 variantpolypeptides, wherein an identified FGF19 variant improves at least onecondition of, for example, a hyperglycemic condition (e.g., diabetes),insulin resistance, hyperinsulinemia, glucose intolerance, metabolicsyndrome, obesity or an undesirable body mass. Additional examples ofmetabolic disorders, diseases and conditions are described hereafter.

In certain embodiments of the methods and models described herein, thesubject (e.g., a test subject) is an animal (e.g., a rodent, or monkey),such as a mouse (e.g., a db/db mouse). Depending on the context in whichthe term is used, a subject can also be a human. In some embodiments,the subject has an increased level of mature FGF19 compared to the levelof mature FGF19 in a sample population, wherein the sample populationcan be any group of members useful as a baseline, reference, etc. Insome embodiments, the increased level of mature FGF19 is due toover-expression.

In some embodiments, the FGF19, a FGF19 surrogate, and/or FGF19 variantis labeled, for example, to facilitate detection, purification and thelike. In certain embodiments, the FGF19, a FGF19 surrogate, and/or FGF19variant is labeled through a covalent bond. The skilled artisan isfamiliar with different types of labels and uses thereof. Labeling ismost frequently effected at the N-terminus and/or C-terminus of apolypeptide, but it can also occur within the polypeptide. The presentdisclosure contemplates the use of any direct and indirect labelingtechniques, which can be carried out in vivo, in vitro, etc.

In the methods and models of the present disclosure, the stepsassociated with determining an indicia of a cancerous condition,disorder or disease, can be performed at any time that can allow thecancerous condition, disorder or disease to manifest itself and thus bedetected. By way of example, the determination can occur more than 3months, more than 20 weeks, more than 6 months, more than 9 months, ormore than 12 months after the aforementioned co-administration steps. Inparticular embodiments, FGF19 is co-administered with the FGF19 variant.

The present disclosure also contemplates a method of antagonizing theoncogenic activity of FGF19. In certain embodiments, provided herein isa method of antagonizing the oncogenic activity of FGF19 in a subject,comprising administering to the subject a therapeutically effectiveamount of a FGF19 variant, thereby antagonizing the oncogenic activityof FGF19 in the subject. In certain embodiments, the subject has ametabolic disorder and/or an indicia of a cancerous condition.

The present disclosure further contemplates a method of preventing ortreating a FGF19-dependent disease, disorder or condition, or a symptomthereof, in a subject, comprising administering to the subject atherapeutically effective amount of a FGF19 variant, wherein thedisease, disorder or condition thereof in the subject. is prevented ortreated. In certain embodiments, there is an improvement in the disease,disorder, condition or symptom thereof in the subject. In certainembodiments, the subject has a metabolic disorder and/or an indicia of acancerous condition. In a specific embodiment, the FGF19-dependentdisease, disorder or condition is a cancer or tumor. In someembodiments, the cancer or tumor is a liver cancer or tumor. In certainembodiments, the cancer or tumor is a colon cancer or tumor. In otherembodiments, the cancer or tumor is a prostate cancer or tumor. In yetother embodiments, the cancer or tumor is a lung cancer or tumor. Incertain embodiments, the subject is a subject in need of prevention ortreatment thereof. In a specific embodiment, the FGF19 variant is apolypeptide comprising or consisting of an amino acid sequence set forthin SEQ ID NO:1 (M70).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the amino acid sequences of mature human FGF19. The aminoacid residues corresponding to the flag epitope are underlined.

FIG. 2 depicts the plasma FGF19 concentrations determined by ELISA indb/db mice five weeks following AAV-mediated gene delivery (GFP as acontrol; FGF19; and/or M70).

FIG. 3 depicts gross hepatic tumor nodule formation in db/db mice aftercontinuous exposure to GFP; FGF19-flag; and/or M70 twenty-four weeksfollowing AAV-mediated gene delivery.

FIG. 4 depicts the effect on body weight, measured prior to injectionand 3-, 5- and 23-weeks post-injection, in db/db mice after continuousexposure to GFP; FGF19-flag; and/or M70 following AAV-mediated genedelivery.

FIG. 5 depicts the effect on glucose concentration, measured prior toinjection and 3-, S- and 23-weeks post-injection, in db/db mice aftercontinuous exposure to GFP; FGF19-flag; and/or M70 followingAAV-mediated gene delivery.

FIGS. 6A-6E depict an AAV-mediated transgene system for studyinghepatocellular tumorigenesis. (A) A diagram of experimental protocol.Mice were given a single injection of 3×10¹¹ genome copies of AAV-FGF19via tail vein when 6-12 week old. Mice were sacrificed 24 or 52 weeklater for liver tumor analysis. ITR, inverted terminal repeat; EF1a,elongation factor 1α promoter. (B) Representative livers of db/db mice24 weeks after administration of AAV-FGF19. Multiple, large, raisedtumors protruding from the hepatic surface were observed in db/db miceexpressing FGF19. No liver tumor was observed in animals injected with acontrol virus (AAV-GFP) in this experiment. Scale bars, 10 mm. (C) Serumlevels of FGF19 were measured by ELISA at 1, 4, 12, and 24 weeks afterAAV administration in db/db mice (n=5). All values represent mean±SEM.(D) Liver tumor multiplicity, size, and scoring in db/db mice expressingFGF19 transgene. Tumors per liver were counted and maximal tumor sizeswere measured. The mean in each group is indicated by horizontal lines(n=15 per group, each dot represents an individual animal). All valuesrepresent mean±SEM. ***p<0.001, *p<0.05 denote significant differencesvs. control group by two-tailed t test. (E) Histological andimmunohistochemical characterization of FGF19-induced liver tumors indb/db mice. The columns are, from top to bottom: hematoxylin and eosin(H & E) staining of liver sections; immunohistochemical detection ofKi-67, PCNA, glutamine synthetase, and β-catenin. FGF19-inducedneoplastic cells are strongly glutamine synthetase-positive. Tumors (T)are outlined by dotted lines. Scale bars, 100 μm.

FIGS. 7A-7H depict M70 is a tumor-free FGF19 variant after continuousexposure in db/db mice for 24 weeks. (A) Alignment of protein sequencesof M70 and FGF19 in the N-terminal region. Mutations introduced into M70are underlined. (B)-(F) Number of tumors per liver (B), liver weight(C), and ratio of liver to body weight (D) of db/db mice expressingFGF19 or M70 for 24 weeks (n=5 per group). Growth curve (E) and serumlevels of transgene expression (F) were also determined. (G)Representative liver sections from db/db mice after 24 weeks oftransgene expression. The liver panel columns are, from top to bottom:hematoxylin and eosin (H & E) staining of liver tissue sections;immunohistochemical detection of Ki-67 and glutamine synthetase. Tumors(T) are outlined by dotted lines. Scale bars, 100 μm. (H) Serum levelsof liver enzymes (ALKP: alkaline phosphatase; ALT, alanineaminotransferase; AST, aspartate aminotransferase; n=5 per group) weremeasured prior to termination of the study. All values representmean±SEM. *p<0.05, **p<0.01, ***p<0.001 denotes significant differencesvs. control group by one-way ANOVA followed by Dunnett's post test. Seealso Tables 3 and 4.

FIGS. 8A-8G depict no liver tumor formation in rasH2 mice treated withM70 for 52 weeks. (A)-(E) Growth curve (A), number of tumors per liver(B), liver weight (C), liver-to-body weight ratios (D), and serum levelsof M70 or FGF19 (E) of rasH2 mice expressing FGF19 or M70 transgenes(n=9 per group) for 52 weeks. (F) Livers were collected 52 weeks afterAAV administration and stained with H & E or anti-glutamine synthetase,a marker for FGF19-induced liver tumors. The sections stained forglutamine synthetase were taken from an area near the paired sectionstained with H & E and showed the same portal (p) and central (c) veins.Tumors (T) are outlined by dotted lines. Scale bars, 100 μm. (G) qRT-PCRanalysis of Ki-67 and AFP expression in the liver. mRNA abundance wasnormalized to GAPDH expression. All values represent mean±SEM. *p<0.05,**p<0.01, ***p<0.001 denotes significant differences vs. control groupby one-way ANOVA followed by Dunnett's post test.

FIGS. 9A-9G depicts M70 binding and activation of FGFR4 in vitro. (A)Biacore SPR assay of the interaction between FGF19 and FGFR4-Fc chimericproteins immobilized on flow cells. Left column shows binding curvesobtained over a range of FGF19 concentrations (15.62-2000 nM at 2 folddilutions), while right column shows the steady state fits of the datafor obtaining K_(D) values. (B) Binding of M70 to FGFR4 by Biacore.Similar procedures to (A) were used. (C) Solid phase binding of M70 orFGF19 to FGFR4-KLB receptor complex. The bound ligands were detectedusing a biotinylated FGF19-specific polyclonal antibody. (D) Relativeluciferase activity after stimulation with M70 or FGF19 in L6 cellstransiently transfected with FGFR4 in the presence or absence of KLB.(E) M70 induces ERK phosphorylation in Hep3B cells. (F) M70 repressedCyp7a1 expression in primary hepatocytes of mouse, rat, and humanorigin. Relative expression of Cyp7a1 mRNA in hepatocytes weredetermined by qRT-PCR and normalized to 18S RNA (mouse and rat) or actin(human) mRNA levels. (G) Repression of hepatic Cyp7a1 expression by M70in mice. 12-week-old db/db mice were injected intraperitoneally withrecombinant M70 or FGF19 protein. Mice were euthanized 4 hours afterdosing and hepatic Cyp7a1 expression was evaluated by qRT-PCR andnormalized to 18S RNA expression. Dose response curves of Cyp7a1repression in mice were shown. All values represent mean±SEM.

FIGS. 10A-10C depict the differential activation of cell signalingpathways by M70 and FGF19 in vivo. (A) Livers were harvested from db/dbmice (n=6 per group) injected intraperitoneally with saline, 1 mg/kgFGF19 or 1 mg/kg M70 proteins 2 hours post injection. Liver lysates wereexamined by western blot for expression and phosphorylation of theindicated proteins. Each lane represents an individual mouse. Rab11serves as a loading control. Note that hepatic STAT3 is activated byFGF19, not M70. (B) Mice treated with FGF19 exhibited elevatedexpression of IL-6 (a STAT3 inducer). Livers were harvested from db/dbmice as in (A). IL-6 mRNA amounts in livers were measured by qRT-PCR andnormalized to GAPDH expression. Results are represented as foldexpression relative to saline-treated animals. Shown are the results for5 separate mice per condition. STAT3 phosphorylation status byimmunoblotting the liver lysates from the same animals is shown in thelower panel. (C) qPCR showing expression of mRNAs for STAT3 target genes(survivin, Bcl-X_(L), and cyclin D1) in rasH2 mice 52 weeks followingadministration of AAV vectors expressing FGF19 or M70 transgenes. Allvalues represent mean±SEM. **p<0.01, ***p<0.001 denotes significantdifferences vs. control group by one-way ANOVA followed by Dunnett'spost test.

FIGS. 11A-11J depicts M70 inhibits FGF19-induced tumor growth in db/dbmice and in xenograft models. (A)-(D) 11 week old db/db mice wereinjected with AAV-FGF19 (3×10¹⁰ genome copies) in the absence orpresence of M70 (3×10¹¹ genome copies). Liver tumor score (A), liverweight (B), ratio of liver to body weight (C) and serum levels oftransgene expression (D) were determined 24 weeks later. *p<0.05 denotessignificant differences vs. control group by one-way ANOVA followed byDunnett's post test; ## p<0.01 denotes significant differences bytwo-tailed t test. (E) Histology of livers of mice expressing FGF19 orco-treated with M70. Liver sections were stained with H & E oranti-glutamine synthetase, a marker for FGF19-induced liver tumors.Tumors (T) are outlined by dotted lines. Scale bars, 100 μm. (F) FGF19is produced and secreted by human cancer cell lines. FGF19 levels inculture supernatant are determined by ELISA. (G-J) M70 inhibits humancancer xenograft tumor growth in vivo. 8 week old athymic nu/nu micewere subcutaneously implanted with 5×10⁶ Huh-7 (n=10) (G) or HCT-116(n=5) (H-J) cells. Mice bearing established tumors of equivalent volumes(˜100 mm³) were randomized into groups and treated with M70 viaAAV-mediated gene delivery. A control virus (GFP) was also included inthe study. Tumor growth was measured over the course of a 15-daytreatment period. The image shows HCT-116 solid tumors dissected at endof the 15-day treatment period (I). Body weight gain of mice bearingHCT-116 tumor xenografts (J) were also determined. ***p<0.001 denotessignificant differences vs. control group by two-way ANOVA followed byBonferroni's post test. All values represent mean±SEM.

FIGS. 12A-12B depict a model of developing a FGF19 variant for treatingFGF19-dependent tumors. (A) Chronic liver injury (cholestasis,cirrhosis, etc.) leads to FGF19 accumulation in the liver. Whileimportant for regulating bile acid synthesis, FGF19 also activatesSTAT3, a key transcription factor in promoting hepatocarcinogenesis.This contributes to tumor initiation, promotion, and progression intoHCC. (B) M70 is an engineered variant of FGF19. As a selectivemodulator, M70 exhibits bias toward certain FGFR4 signaling pathways(i.e. pERK and Cyp7a1) to the relative exclusion of others (i.e.,tumor). Moreover, M70 can inhibit the growth of tumors that aredependent on FGF19.

FIGS. 13A-13B depicts M70 delays tumor growth in a CT26 colon cancersyngenic mouse model. (A) M70 delays CT26 tumor growth followingadministration of 10 mg/kg doses. (B) M70 delays CT26 tumor growthfollowing administration of 3 mg/kg doses. The p-values were determinedby two-way ANOVA vs. vehicle-treated mice. *** p<0.001; ** p<0.01.

FIGS. 14A-14B depicts M70 reduces body weight in a CT26 colon cancersyngenic mouse model. (A) M70 reduces body weight followingadministration of 10 mg/kg doses. (B) M70 reduces body weight followingadministration of 3 mg/kg doses. The p-values were determined by two-wayANOVA vs. vehicle-treated mice. *** p<0.001; ** p<0.01.

DETAILED DESCRIPTION

Before the present disclosure is further described, it is to beunderstood that the disclosure is not limited to the particularembodiments set forth herein, and it is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

Overview

The present disclosure contemplates the identification of agents, andcompositions thereof, using the models and methods described herein. Themodels and associated methods provide an accurate, efficient methodologyfor the identification of agents that do not induce cancerous conditions(e.g., hepatocellular carcinoma). In certain embodiments, the models andmethods provided herein are useful in identifying agents that antagonizethe oncogenic activity of FGF19. In certain embodiments, such agentshave therapeutic utility in the treatment and/or prevention of variousdiseases, disorders and conditions, and/or the symptoms thereof,pertaining to, for example, glucose metabolism disorders and/or bodyweight disorders. By way of example, but not limitation, the agents, andcompositions thereof, can be used for the treatment and/or prevention oftype 2 diabetes, insulin resistance and diseases, disorders andconditions characterized by insulin resistance, decreased insulinproduction, hyperglycemia, metabolic syndrome, or obesity. Such agentsare also useful in the prevention or treatment a FGF19-dependentdisease, disorder or condition, or a symptom thereof.

The models and associated methods described herein are useful inidentifying agents (e.g., polypeptides and antibodies) that neitherinduce nor exacerbate the cancer-related effects of FGF19 (e.g., HCC).As described in detail hereafter, particular embodiments contemplate theuse of the models and methods to determine whether a FGF19 variantpolypeptide having favorable metabolic characteristics will also possessa desirable “cancer-related” profile. Also provided are methods ofantagonizing the oncogenic activity of FGF19 in a subject and, incertain embodiments, methods of preventing or treating a FGF19-dependentdisease, disorder or condition, or a symptom thereof. In certainembodiments, the FGF19 dependent disease, disorder or condition is acancer or tumor, such as a liver, colon, prostate or lung cancer ortumor.

Definitions

The terms “patient” or “subject” are used interchangeably to refer to ahuman or a non-human animal (e.g., a mammal).

The terms “treat”, “treating”, treatment” and the like refer to a courseof action (such as administering a polypeptide or a pharmaceuticalcomposition comprising a polypeptide) initiated after a disease,disorder or condition, or a symptom thereof, has been diagnosed,observed, and the like so as to eliminate, reduce, suppress, mitigate,or ameliorate, either temporarily or permanently, at least one of theunderlying causes of a disease, disorder, or condition afflicting asubject, or at least one of the symptoms associated with a disease,disorder, condition afflicting a subject. Thus, treatment includesinhibiting (i.e., arresting the development or further development ofthe disease, disorder or condition or clinical symptoms associationtherewith) an active disease (e.g., so as to decrease the level ofinsulin and/or glucose in the bloodstream, to increase glucose toleranceso as to minimize fluctuation of glucose levels, and/or so as to protectagainst diseases caused by disruption of glucose homeostasis).

The term “in need of treatment” as used herein refers to a judgment madeby a physician or other medical professional that a subject requires orwill benefit from treatment.

The terms “prevent”, “preventing”, “prevention” and the like refer to acourse of action (such as administering a polypeptide or apharmaceutical composition comprising a polypeptide) initiated in amanner (e.g., prior to the onset of a disease, disorder, condition orsymptom thereof) so as to prevent, suppress, inhibit or reduce, eithertemporarily or permanently, a subject's risk of developing a disease,disorder, condition or the like (as determined by, for example, theabsence of clinical symptoms) or delaying the onset thereof, generallyin the context of a subject predisposed to having a particular disease,disorder or condition. In certain instances, the terms also refer toslowing the progression of the disease, disorder or condition orinhibiting progression thereof to a harmful or otherwise undesiredstate.

The term “in need of prevention” as used herein refers to a judgmentmade by a physician or other medical professional that a subjectrequires or will benefit from preventative care.

The phrase “therapeutically effective amount” refers to theadministration of an agent to a subject, either alone or as a part of apharmaceutical composition and either in a single dose or as part of aseries of doses, in an amount that is capable of having any detectable,positive effect on any symptom, aspect, or characteristics of a disease,disorder or condition when administered to a patient. Thetherapeutically effective amount can be ascertained by measuringrelevant physiological effects. In the case of a hyperglycemiccondition, a lowering or reduction of blood glucose or an improvement inglucose tolerance test can be used to determine whether the amount of anagent is effective to treat the hyperglycemic condition. For example, atherapeutically effective amount is an amount sufficient to reduce ordecrease any level (e.g., a baseline level) of fasting plasma glucose(FPG), wherein, for example, the amount is sufficient to reduce a FPGlevel greater than 200 mg/dl to less than 200 mg/dl, wherein the amountis sufficient to reduce a FPG level between 175 mg/dl and 200 mg/dl toless than the starting level, wherein the amount is sufficient to reducea FPG level between 150 mg/dl and 175 mg/dl to less than the startinglevel, wherein the amount is sufficient to reduce a FPG level between125 mg/dl and 150 mg/dl to less than the starting level, and so on(e.g., reducing FPG levels to less than 125 mg/dl, to less than 120mg/dl, to less than 115 mg/dl, to less than 110 mg/dl, etc.). Moreover,in the case of HbAIc levels, the effective amount is an amountsufficient to reduce or decrease levels by more than about 10% to 9%, bymore than about 9% to 8%, by more than about 8% to 7%, by more thanabout 7% to 6%, by more than about 6% to 5%, and so on. Moreparticularly, a reduction or decrease of HbAIc levels by about 0.1%,0.25%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 3%, 4%, 5%,10%, 20%, 30%, 33%, 35%, 40%, 45%, 50%, or more is contemplated by thepresent disclosure. The therapeutically effective amount can be adjustedin connection with the dosing regimen and diagnostic analysis of thesubject's condition and the like.

The phrase “in a sufficient amount to effect a change” means that thereis a detectable difference between a level of an indicator measuredbefore (e.g., a baseline level) and after administration of a particulartherapy. Indicators include any objective parameter (e.g., level ofglucose or insulin) or subjective parameter (e.g., a subject's feelingof well-being).

The phrase “glucose tolerance”, as used herein, refers to the ability ofa subject to control the level of plasma glucose and/or plasma insulinwhen glucose intake fluctuates. For example, glucose toleranceencompasses the subject's ability to reduce, within about 120 minutes,the level of plasma glucose back to a level determined before the intakeof glucose.

Broadly speaking, the terms “diabetes” and “diabetic” refer to aprogressive disease of carbohydrate metabolism involving inadequateproduction or utilization of insulin, frequently characterized byhyperglycemia and glycosuria. The terms “pre-diabetes” and“pre-diabetic” refer to a state wherein a subject does not have thecharacteristics, symptoms and the like typically observed in diabetes,but does have characteristics, symptoms and the like that, if leftuntreated, can progress to diabetes. The presence of these conditionscan be determined using, for example, either the fasting plasma glucose(FPG) test or the oral glucose tolerance test (OGTT). Both usuallyrequire a subject to fast for at least 8 hours prior to initiating thetest. In the FPG test, a subject's blood glucose is measured after theconclusion of the fasting; generally, the subject fasts overnight andthe blood glucose is measured in the morning before the subject eats. Ahealthy subject would generally have a FPG concentration between about90 and about 100 mg/dl, a subject with “pre-diabetes” would generallyhave a FPG concentration between about 100 and about 125 mg/dl, and asubject with “diabetes” would generally have a FPG level above about 126mg/dl. In the OGTT, a subject's blood glucose is measured after fastingand again two hours after drinking a glucose-rich beverage. Two hoursafter consumption of the glucose-rich beverage, a healthy subjectgenerally has a blood glucose concentration below about 140 mg/dl, apre-diabetic subject generally has a blood glucose concentration about140 to about 199 mg/dl, and a diabetic subject generally has a bloodglucose concentration about 200 mg/dl or above. While the aforementionedglycemic values pertain to human subjects, normoglycemia, moderatehyperglycemia and overt hyperglycemia are scaled differently in murinesubjects. A healthy murine subject after a four-hour fast wouldgenerally have a FPG concentration between about 100 and about 150mg/dl, a murine subject with “pre-diabetes” would generally have a FPGconcentration between about 175 and about 250 mg/dl and a murine subjectwith “diabetes” would generally have a FPG concentration above about 250mg/dl.

The term “insulin resistance” as used herein refers to a condition wherea normal amount of insulin is unable to produce a normal physiologicalor molecular response. In some cases, a hyper-physiological amount ofinsulin, either endogenously produced or exogenously administered, isable to overcome the insulin resistance, in whole or in part, andproduce a biologic response.

The term “metabolic syndrome” refers to an associated cluster of traitsthat includes, but is not limited to, hyperinsulinemia, abnormal glucosetolerance, obesity, redistribution of fat to the abdominal or upper bodycompartment, hypertension, dysfibrinolysis, and dyslipidemiacharacterized by high triglycerides, low high density lipoprotein(HDL)-cholesterol, and high small dense low density lipoprotein (LDL)particles. Subjects having metabolic syndrome are at risk fordevelopment of type 2 diabetes and/or other disorders (e.g.,atherosclerosis).

The phrase “glucose metabolism disorder” encompasses any disordercharacterized by a clinical symptom or a combination of clinicalsymptoms that is associated with an elevated level of glucose and/or anelevated level of insulin in a subject relative to a healthy individual.Elevated levels of glucose and/or insulin can be manifested in thefollowing diseases, disorders and conditions: hyperglycemia, type IIdiabetes, gestational diabetes, type I diabetes, insulin resistance,impaired glucose tolerance, hyperinsulinemia, impaired glucosemetabolism, pre-diabetes, other metabolic disorders (such as metabolicsyndrome, which is also referred to as syndrome X), and obesity, amongothers. The polypeptides of the present disclosure, and compositionsthereof, can be used, for example, to achieve and/or maintain glucosehomeostasis, e.g., to reduce glucose level in the bloodstream and/or toreduce insulin level to a range found in a healthy subject.

The term “hyperglycemia”, as used herein, refers to a condition in whichan elevated amount of glucose circulates in the blood plasma of asubject relative to a healthy individual. Hyperglycemia can be diagnosedusing methods known in the art, including measurement of fasting bloodglucose levels as described herein.

The term “hyperinsulinemia”, as used herein, refers to a condition inwhich there are elevated levels of circulating insulin when,concomitantly, blood glucose levels are either elevated or normal.Hyperinsulinemia can be caused by insulin resistance which is associatedwith dyslipidemia, such as high triglycerides, high cholesterol, highlow-density lipoprotein (LDL) and low high-density lipoprotein (HDL);high uric acids levels; polycystic ovary syndrome; type II diabetes andobesity. Hyperinsulinemia can be diagnosed as having a plasma insulinlevel higher than about 2 μU/mL.

As used herein, the phrase “body weight disorder” refers to conditionsassociated with excessive body weight and/or enhanced appetite. Variousparameters are used to determine whether a subject is overweightcompared to a reference healthy individual, including the subject's age,height, sex and health status. For example, a subject can be consideredoverweight or obese by assessment of the subject's Body Mass Index(BMI), which is calculated by dividing a subject's weight in kilogramsby the subject's height in meters squared. An adult having a BMI in therange of ˜18.5 to ˜24.9 kg/m² is considered to have a normal weight; anadult having a BMI between ˜25 and ˜29.9 kg/m² can be consideredoverweight (pre-obese); and an adult having a BMI of ˜30 kg/m² or highercan be considered obese. Enhanced appetite frequently contributes toexcessive body weight. There are several conditions associated withenhanced appetite, including, for example, night eating syndrome, whichis characterized by morning anorexia and evening polyphagia oftenassociated with insomnia, but which can be related to injury to thehypothalamus.

The terms “polypeptide,” “peptide,” and “protein”, used interchangeablyherein, refer to a polymeric form of amino acids of any length, whichcan include genetically coded and non-genetically coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified polypeptide backbones. The terms includefusion proteins, including, but not limited to, fusion proteins with aheterologous amino acid sequence, fusion proteins with heterologous andhomologous leader sequences, with or without N-terminus methionineresidues; immunologically tagged proteins; and the like. It will beappreciated that throughout this disclosure reference is made to aminoacids according to the single letter or three letter codes.

As used herein, the term “variant” encompasses naturally-occurringvariants (e.g., homologs and allelic variants) andnon-naturally-occurring variants (e.g., muteins). Naturally-occurringvariants include homologs, i.e., nucleic acids and polypeptides thatdiffer in nucleotide or amino acid sequence, respectively, from onespecies to another. Naturally-occurring variants include allelicvariants, i.e., nucleic acids and polypeptides that differ in nucleotideor amino acid sequence, respectively, from one individual to anotherwithin a species. Non-naturally-occurring variants include nucleic acidsand polypeptides that comprise a change in nucleotide or amino acidsequence, respectively, where the change in sequence is artificiallyintroduced, e.g., the change is generated in the laboratory or otherfacility by human intervention (“hand of man”).

The term “native”, in reference to FGF19, refers to biologically active,naturally-occurring FGF19, including biologically active,naturally-occurring FGF19 variants. The term includes the 194 amino acidhuman FGF19 mature sequence.

The terms “label”, “labeling” and the like, when use in the context of apolypeptide or nucleic acid (or antibody, as appropriate) of the presentdisclosure are meant to refer broadly to any means useful in, forexample, polypeptide purification, identification, isolation andsynthesis. Labels are generally covalently bound to the polypeptide ofinterest and can be introduced in any manner known in the art, includingattachment to a mature polypeptide (generally at the N- or C-terminus),incorporation during solid-phase peptide synthesis, or throughrecombinant means. Examples include, but are not limited to,fluorescence, biotinylation, and radioactive isotopes. Polypeptide andnucleic acid molecules can be labeled by both in vitro and in vivomethods. Labeling reagents and kits can be obtained from a number ofcommercial sources (e.g., Thermo Fischer Scientific, Rockford, Ill.; andMolecular Probes/Life Technologies; Grand Island, N.Y.).

As used herein, the terms “FLAG-tag”, “FLAG octapeptide”, and the likerefer to an eight amino acid (DYKDDDDK) (SEQ ID NO:2) peptide tag(label) that can be added to a polypeptide using recombinant DNAtechniques. Antibodies to the FLAG component of the polypeptide can beused for, for example, affinity chromatography and cellular localizationstudies by immunofluorescence or detection by SDS PAGE proteinelectrophoresis. A FLAG-tag can be used in conjunction with otheraffinity tags (e.g., a polyhistidine tag (His-tag) or myc-tag), and itcan be fused to the C-terminus or the N-terminus of a polypeptide.

The term “muteins” as used herein refers broadly to mutated recombinantproteins, i.e., a polypeptide comprising an artificially introducedchange in amino acid sequence, e.g., a change in amino acid sequencegenerated in the laboratory or other facility by human intervention(“hand of man”). These proteins usually carry single or multiple aminoacid substitutions and are frequently derived from cloned genes thathave been subjected to site-directed or random mutagenesis, or fromcompletely synthetic genes.

As used herein in reference to native human FGF19 or a FGF19 mutein, theterms “modified”, “modification” and the like refer to one or morechanges that enhance a desired property of human FGF19, anaturally-occurring FGF19 variant, or a FGF19 mutein, wherein thechange(s) does not alter the primary amino acid sequence of the FGF19.Such desired properties include, for example, enhancing solubility,prolonging the circulation half-life, increasing the stability, reducingthe clearance, altering the immunogenicity or allergenicity, improvingaspects of manufacturability (e.g., cost and efficiency), and enablingthe raising of particular antibodies (e.g., by introduction of uniqueepitopes) for use in detection assays. Changes to human FGF19, anaturally-occurring FGF19 variant, or a FGF19 mutein that can be carriedout include, but are not limited to, pegylation (covalent attachment ofone or more molecules of polyethylene glycol (PEG), or derivativesthereof); glycosylation (e.g., N-glycosylation), polysialylation andhesylation; albumin fusion; albumin binding through, for example, aconjugated fatty acid chain (acylation); Fc-fusion; and fusion with aPEG mimetic. Some particular embodiments entail modifications involvingpolyethylene glycol, other particular embodiments entail modificationsinvolving albumin, and still other particular modifications entailmodifications involving glycosylation.

The terms “DNA”, “nucleic acid”, “nucleic acid molecule”,“polynucleotide” and the like are used interchangeably herein to referto a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Non-limiting examples of polynucleotides include linear and circularnucleic acids, messenger RNA (mRNA), complementary DNA (cDNA),recombinant polynucleotides, vectors, probes, primers and the like.

The term “probe” refers to a fragment of DNA or RNA corresponding to agene or sequence of interest, wherein the fragment has been labeledradioactively (e.g., by incorporating ³²P or ³⁵S) or with some otherdetectable molecule, such as biotin, digoxygen or fluorescein. Asstretches of DNA or RNA with complementary sequences will hybridize, aprobe can be used, for example, to label viral plaques, bacterialcolonies or bands on a gel that contain the gene of interest. A probecan be cloned DNA or it can be a synthetic DNA strand; the latter can beused to obtain a cDNA or genomic clone from an isolated protein by, forexample, microsequencing a portion of the protein, deducing the nucleicacid sequence encoding the protein, synthesizing an oligonucleotidecarrying that sequence, radiolabeling the sequence and using it as aprobe to screen a cDNA library or a genomic library.

The term “heterologous” refers to two components that are defined bystructures derived from different sources. For example, in the contextof a polypeptide, a “heterologous” polypeptide can include operablylinked amino acid sequences that are derived from differentpolypeptides. Similarly, in the context of a polynucleotide encoding achimeric polypeptide, a “heterologous” polynucleotide can includeoperably linked nucleic acid sequences that can be derived fromdifferent genes. Exemplary “heterologous” nucleic acids includeexpression constructs in which a nucleic acid comprising a codingsequence is operably linked to a regulatory element (e.g., a promoter)that is from a genetic origin different from that of the coding sequence(e.g., to provide for expression in a host cell of interest, which canbe of different genetic origin than the promoter, the coding sequence orboth). In the context of recombinant cells, “heterologous” can refer tothe presence of a nucleic acid (or gene product, such as a polypeptide)that is of a different genetic origin than the host cell in which it ispresent.

The term “operably linked” refers to linkage between molecules toprovide a desired function. For example, “operably linked” in thecontext of nucleic acids refers to a functional linkage between nucleicacid sequences. By way of example, a nucleic acid expression controlsequence (such as a promoter, signal sequence, or array of transcriptionfactor binding sites) can be operably linked to a second polynucleotide,wherein the expression control sequence affects transcription and/ortranslation of the second polynucleotide. In the context of apolypeptide, “operably linked” refers to a functional linkage betweenamino acid sequences (e.g., different domains) to provide for adescribed activity of the polypeptide.

As used herein in the context of the structure of a polypeptide,“N-terminus” (or “amino terminus”) and “C-terminus” (or “carboxylterminus”) refer to the extreme amino and carboxyl ends of thepolypeptide, respectively, while the terms “N-terminal” and “C-terminal”refer to relative positions in the amino acid sequence of thepolypeptide toward the N-terminus and the C-terminus, respectively, andcan include the residues at the N-terminus and C-terminus, respectively.“Immediately N-terminal” or “immediately C-terminal” refers to aposition of a first amino acid residue relative to a second amino acidresidue where the first and second amino acid residues are covalentlybound to provide a contiguous amino acid sequence.

“Derived from”, in the context of an amino acid sequence orpolynucleotide sequence (e.g., an amino acid sequence “derived from” aFGF19 polypeptide), is meant to indicate that the polypeptide or nucleicacid has a sequence that is based on that of a reference polypeptide ornucleic acid (e.g., a naturally occurring FGF19 polypeptide or aFGF19-encoding nucleic acid), and is not meant to be limiting as to thesource or method in which the protein or nucleic acid is made. By way ofexample, the term “derived from” includes homologues or variants ofreference amino acid or DNA sequences.

In the context of a polypeptide, the term “isolated” refers to apolypeptide of interest that, if naturally occurring, is in anenvironment different from that in which it can naturally occur.“Isolated” is meant to include polypeptides that are within samples thatare substantially enriched for the polypeptide of interest and/or inwhich the polypeptide of interest is partially or substantiallypurified. Where the polypeptide is not naturally occurring, “isolated”indicates the polypeptide has been separated from an environment inwhich it was made by either synthetic or recombinant means.

“Enriched” means that a sample is non-naturally manipulated (e.g., by ascientist or a clinician) so that a polypeptide of interest is presentin a) a greater concentration (e.g., at least 3-fold greater, at least4-fold greater, at least 8-fold greater, at least 64-fold greater, ormore) than the concentration of the polypeptide in the starting sample,such as a biological sample (e.g., a sample in which the polypeptidenaturally occurs or in which it is present after administration), or b)a concentration greater than the environment in which the polypeptidewas made (e.g., as in a bacterial cell).

“Substantially pure” indicates that a component (e.g., a polypeptide)makes up greater than about 50% of the total content of the composition,and typically greater than about 60% of the total polypeptide content.More typically, “substantially pure” refers to compositions in which atleast 75%, at least 85%, at least 90% or more of the total compositionis the component of interest. In some cases, the polypeptide will makeup greater than about 90%, or greater than about 95% of the totalcontent of the composition.

The terms “assaying” and “measuring” and grammatical variations thereofare used interchangeably herein and refer to either qualitative orquantitative determinations, or both qualitative and quantitativedeterminations. When the terms are used in reference to detection, anymeans of assessing the relative amount is contemplated, including thevarious methods set forth herein and known in the art. For example, geneexpression can be assayed or measured by a Northern blot, Western blot,immunoprecipitation assay, or by measuring activity, function or amountof the expressed protein.

The terms “antibodies” (Abs) and “immunoglobulins” (Igs) refer toglycoproteins having the same structural characteristics. Whileantibodies exhibit binding specificity to a specific antigen,immunoglobulins include both antibodies and other antibody-likemolecules which lack antigen specificity.

The term “monoclonal antibody” refers to an antibody obtained from apopulation of substantially homogeneous antibodies, that is, theindividual antibodies comprising the population are identical except forpossible naturally occurring mutations that can be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. In contrast to polyclonal antibodypreparations, which can include different antibodies directed againstdifferent determinants (epitopes), each monoclonal antibody is directedagainst a single determinant on the antigen.

In the context of an antibody, the term “isolated” refers to an antibodythat has been separated and/or recovered from contaminant components ofits natural environment; such contaminant components include materialswhich might interfere with diagnostic or therapeutic uses for theantibody, and can include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes.

As used herein, the term “FGF19-dependent” and similar terms, as used inthe context of a disease, disorder or condition, refers to a disease,disorder or other condition that is caused all, or in part, by theexpression of FGF19. In certain embodiments, the expression of FGF19 isamplified as compared to a control. In some embodiments, the expressionof FGF19 is amplified 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, or any numericalrange thereof. In some embodiments, the amplified expression of FGF19directly results in the disease, disorder or condition, or a symptomthereof. In other embodiments, the amplified expression of FGF19indirectly results in the disease disorder or condition, or a symptomthereof.

Fibroblast Growth Factor 19 (FGF19)

Fibroblast growth factors (FGFs) are a family of growth factors thatplay key roles in cellular proliferation and differentiation. Twenty-twomembers of the FGF family have been identified in humans, all of whichare structurally-related signaling molecules. The FGF19 subfamily ofFGFs consists of human FGF21, FGF23 and FGF19 and mouse FGF15.

The physiological effects of FGF family members are the result ofheparin-dependent binding to one or more members of the FGF receptortyrosine kinase (FGFR) family, which includes four members (FGFR1,FGFR2, FGFR3 and FGFR4), each having a tyrosine kinase domain. Inaddition, each of FGFR1, FGFR2 and FGFR3 also has two splice variantsdesignated as “b” and “c” variants (i.e., FGFR1b, FGFR2b, FGFR3b,FGFR1c, FGFR2c and FGFR3c).

FGF19 targets and has effects on both adipocytes and hepatocytes. Micetreated with recombinant human FGF19, despite being on a high-fat diet,show increased metabolic rates, increased lipid oxidation, a lowerrespiratory quotient, and weight loss. The metabolic effects of FGF19occur via its binding to the FGFR1c, FGFR2c and FGFR3c receptors, ofwhich the binding to FGFR1c and FGFR2c are the most significant. FGF19binding to these receptors requires the co-receptor Klotho-β (KLB).

FGF19 has also been shown to regulate bile production by the liver.Thus, FGF19-like agents can play an important role in bile acidhomeostasis. Results suggest that FGF19-regulated liver bile acidmetabolism could be independent of its glucose-lowering effect.

As alluded to elsewhere herein, use of gastric bypass surgery for thetreatment of diabetes has been shown to completely and persistently curetype II diabetes in most patients. This “bariatric effect” is evidentonly days after surgery and long before significant weight loss isachieved. FGF19 levels increase after bariatric surgery, and it can beresponsible for the bariatric effect.

FGF19 is expressed as a 216 amino acid polypeptide comprising a 22residue signal peptide (GenBank: AAQ88669.1). Mature human FGF19(wild-type) is a 194 amino acid polypeptide comprising the followingamino acid sequence:

(SEQ ID NO: 3) RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKEIRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK.

FGF19 and Hepatocellular Carcinoma.

As described herein, FGF19 is associated with the induction of cancer,particularly HCC, the most common type of liver cancer. In accordancewith certain aspects, there are provided methods and models ofidentifying a polypeptide, or a subsequence, variant or modified formthereof, as set forth herein, having a desired metabolic activity (e.g.,glucose lowering activity) but lacking or without substantial HCCactivity. Various metabolic disorders and associated methods (e.g.,methods of measuring glucose levels), along with methods of detectingcancers, are described elsewhere herein and are known in the art.

Various methodologies can be used in the screening and diagnosis of HCCand are well known to the skilled artisan. Indicators for HCC include,but are not limited to, detection of a tumor maker, such as elevatedalpha-fetoprotein (AFP) or des-gamma carboxyprothrombin (DCP) levels. Anumber of different scanning and imaging techniques are also available,including ultrasound, CT scans and MRI. In relation to certainembodiments of the methods and models provided herein, evaluation ofwhether a polypeptide (e.g., a candidate polypeptide) exhibits evidenceof inducing HCC is determined in vivo by, for example, quantifying HCCnodule formation in an animal model (e.g., a db/db mouse model)administered a polypeptide, compared to HCC nodule formation induced bywild-type FGF19. Macroscopically, HCC can be nodular, whereas the tumornodules (which are frequently round-to-oval, grey or green, wellcircumscribed but not encapsulated) appear as either one large mass ormultiple smaller masses. Alternatively, HCC can be present as aninfiltrative tumor which is diffuse and poorly circumscribed andfrequently infiltrates the portal veins. Risk factors for HCC includetype 2 diabetes (often exacerbated by obesity). The risk of HCC in type2 diabetics is greater (from ˜2.5 to ˜7 times the non-diabetic risk)depending on the duration of diabetes and treatment protocol.

Pathological assessment of hepatic tissue samples is generally performedafter the results of one or more of the aforementioned methodologiesindicate the likely presence of HCC. Thus, certain embodiments of themethods provided herein further include assessing a hepatic tissuesample from an in vivo animal model useful in HCC studies in order todetermine whether a polypeptide sequence exhibits evidence of inducingHCC. In certain embodiments, the in vivo animal model is a db/db mousemodel. By microscopic assessment, a pathologist can determine whetherone of the four general architectural and cytological types (patterns)of HCC are present (i.e., fibrolamellar, pseudoglandular (adenoid),pleomorphic (giant cell) and clear cell).

Methods and Models for Identifying FGF19 Variant Polypeptides HavingDesired Characteristics.

FGF19 variant polypeptides and other agents that mimic, at least in somerespects, the activity of FGF19 are described in both the scientific andpatent literature. See, e.g., Wu et al., PLos One, 6:e17868 (Mar. 11,2001); U.S. Pat. No. 8,324,160; and US Publ. Nos. 2011/0195895;2011/0207912 and 2011/0104152. Though not intended to be limiting in anyway, candidate FGF19 variant sequences include polypeptides having aWGDPI (SEQ ID NO:4) sequence motif corresponding to the WGDPI sequenceof amino acids 16-20 of FGF19 (SEQ ID NO:3). A particular polypeptidecontemplated herein has the following amino acid sequence:

(M70, SEQ ID NO: 1) MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKIIRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK.

In other embodiments, a FGF19 variant comprises or consists of an aminoacid sequence set forth as:

(M5, SEQ ID NO: 5)RHPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS PSFEK;(M6, SEQ ID NO: 6)RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKEIRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K;(M7, SEQ ID NO: 7)RPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVR SPSFEK;(M14, SEQ ID NO: 8)RHPIPDSSPHVHYGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVR SPSFEK;(M15, SEQ ID NO: 9)RPLAFSDAGPHVHYGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKEIRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEA VRSPSFEK;(M32, SEQ ID NO: 10)RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS PSFEK;(M36, SEQ ID NO: 11)RHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS PSFEK;(M43, SEQ ID NO: 12)RPLAFSDAGPHVHYGGDIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAV RSPSFEK;(M50, SEQ ID NO: 13)RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS PSFEK;(M52, SEQ ID NO: 14)RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K;(M53, SEQ ID NO: 15)MDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K;(M67, SEQ ID NO: 16)RPLAFSDAGPHVWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVR SPSFEK;(M68, SEQ ID NO: 17)RPLAFSDAGPHVHYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEA VRSPSFEK;(M69, SEQ ID NO: 18)RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKEIRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSP SFEK;(M70, SEQ ID NO: 1 or 19)MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS PSFEK;(M75, SEQ ID NO: 20)RVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;(M76, SEQ ID NO: 21)RGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;(M77, SEQ ID NO: 22)RRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;(M83, SEQ ID NO: 23)RPLAFSDAAPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M84, SEQ ID NO: 24)RPLAFSDAGAHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M140, SEQ ID NO: 25)RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIREDGYNVYRSEKEIRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (M144 (M5-R), SEQ ID NO: 26)HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSP SFEK;(M145 (M6-R), SEQ ID NO: 27)DSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K;(M146 (M50-R), SEQ ID NO: 28)HPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSP SFEK;(M160, SEQ ID NO: 29)RPLAFSDAGPHVHYGWGDPIRQRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;or a subsequence or fragment thereof of any of the foregoing peptidesequences. In certain embodiments of any of the foregoing peptidesequences, the N-terminal R residue is deleted.

As previously described, one aspect of the present disclosurecontemplates a method for determining whether a test subject having ametabolic disorder is a candidate for treatment with a FGF19 variant,the method comprising: (a) providing a test subject having an indicia ofa cancerous condition, the subject having a metabolic disorder, (b)co-administering FGF19 or a FGF19 surrogate, and a FGF19 variant to thetest subject, wherein the amount of the FGF19 or the FGF19 surrogateadministered to the test subject is sufficient to induce a cancerouscondition in a reference population, and (c) determining whether anindicia of a cancerous condition is enhanced in the test subject;wherein the absence of enhancement of an indicia of a cancerouscondition indicates that the test subject is a candidate for treatmentwith a FGF19 variant.

Another aspect of the present disclosure contemplates a method fordetermining whether a test subject having a metabolic disorder is acandidate for treatment with a FGF19 variant, the method comprising: (a)providing a test subject having an indicia of a cancerous condition, thetest subject having a metabolic disorder, (b) co-administering FGF19 ora FGF19 surrogate, and a FGF19 variant to the test subject, wherein theamount of the FGF19 or the FGF19 surrogate is administered to the testsubject is sufficient to induce a cancerous condition in a referencepopulation, and (c) determining whether an indicia of a cancerouscondition is reduced in the test subject; wherein the reduction of anindicia of a cancerous condition indicates that the test subject is acandidate for treatment with a FGF19 variant.

The present disclosure also contemplates a method for determiningwhether a FGF19 variant is a candidate for treating a test subjecthaving a metabolic disorder, the method comprising: (a) co-administeringFGF19 or a FGF19 surrogate, and the FGF19 variant to the test subjecthaving a metabolic disorder, wherein the amount of the FGF19 or theFGF19 surrogate administered to the test subject is sufficient to inducea cancerous condition in a reference population, and (b) determiningwhether an indicia of a cancerous condition is observed in the testsubject; wherein the absence of an indicia of a cancerous conditionindicates that the FGF19 variant is a candidate for treatment of thetest subject.

A further embodiment contemplated herein is drawn to a method fordetermining whether a FGF19 variant is a candidate for treating a testsubject having a metabolic disorder, the method comprising: (a)providing a test subject having a metabolic disorder, the test subjecthaving an indicia of a cancerous condition, (b) co-administering FGF19or a FGF19 surrogate, and a FGF19 variant to the test subject, whereinthe amount of the FGF19 or the FGF19 surrogate is administered to thetest subject is sufficient to exacerbate a cancerous condition in areference population, and (c) determining whether an indicia of acancerous condition is enhanced in the test subject; wherein the absenceof exacerbation of an indicia of a cancerous condition indicates thatthe FGF19 variant is a candidate for treatment of the test subject. Inparticular embodiments, one or more indicia of a cancerous condition arereduced in the test subject.

In certain embodiments of the methods provided herein, the FGF19 variantis selected from the group consisting of M5, M6, M7, M14, M15, M32, M36,M43, M52, M53, M67, M68, M69, M70, M75, M76, M77, M83, M84, M140, M144,M145, M146 and M160. In one embodiment, the FGF19 variant is M5. Inanother embodiment, the FGF19 variant is M6. In some embodiments, theFGF19 variant is M7. In one embodiment, the FGF19 variant is M14. Inanother embodiment, the FGF19 variant is M15. In other embodiments, theFGF19 variant is M32. In one embodiment, the FGF19 variant is M36. Inanother embodiment, the FGF19 variant is M43. In other embodiments, theFGF19 variant is M52. In yet other embodiment, the FGF19 variant is M53.In some embodiments, the FGF19 variant is M67. In one embodiment, theFGF19 variant is M68. In another embodiment, the FGF19 variant is M69.In some embodiments, the FGF19 variant is M70. In one embodiment, theFGF19 variant is M75. In another embodiment, the FGF19 variant is M76.In other embodiments, the FGF19 variant is M77. In yet otherembodiments, the FGF19 variant is M83. In one embodiment, the FGF19variant is M84. In another embodiment, the FGF19 variant is M140. Inother embodiments, the FGF19 variant is M144. In yet other embodiments,the FGF19 variant is M145. In one embodiment, the FGF19 variant is M146.In some embodiments, the FGF19 variant is M160. In other embodiments,any combination of two or more of the foregoing FGF19 variants is alsocontemplated.

In some embodiments of the methods provided herein, the FGF19 variantcomprises an amino acid sequence set forth in any one of SEQ IDNOS:5-29; or a subsequence or fragment thereof. In certain embodiments,the N-terminal R residue is deleted. In some embodiments, the FGF19variant comprises SEQ ID NO:5. In other embodiments, the FGF19 variantcomprises SEQ ID NO:6. In one embodiment, the FGF19 variant comprisesSEQ ID NO:7. In other embodiments, the FGF19 variant comprises SEQ IDNO:8. In another embodiment, the FGF19 variant comprises SEQ ID NO:9. Insome embodiments, the FGF19 variant comprises SEQ ID NO:10. In otherembodiments, the FGF19 variant comprises SEQ ID NO:11. In anotherembodiment, the FGF19 variant comprises SEQ ID NO:12. In someembodiments, the FGF19 variant comprises SEQ ID NO:13. In otherembodiments, the FGF19 variant comprises SEQ ID NO:14. In oneembodiment, the FGF19 variant comprises SEQ ID NO:15. In anotherembodiment, the FGF19 variant comprises SEQ ID NO:16. In someembodiments, the FGF19 variant comprises SEQ ID NO:17. In otherembodiments, the FGF19 variant comprises SEQ ID NO:18. In yet otherembodiments, the FGF19 variant comprises SEQ ID NO:19. In someembodiments, the FGF19 variant comprises SEQ ID NO:20. In oneembodiment, the FGF19 variant comprises SEQ ID NO:21. In someembodiments, the FGF19 variant comprises SEQ ID NO:22. In otherembodiments, the FGF19 variant comprises SEQ ID NO:23. In anotherembodiment, the FGF19 variant comprises SEQ ID NO:24. In someembodiments, the FGF19 variant comprises SEQ ID NO:25. In otherembodiments, the FGF19 variant comprises SEQ ID NO:26. In yet otherembodiments, the FGF19 variant comprises SEQ ID NO:27. In someembodiments, the FGF19 variant comprises SEQ ID NO:28. In otherembodiments, the FGF19 variant comprises SEQ ID NO:29. In certainembodiments, the FGF19 variant comprises any one of the foregoingsequences, wherein the N-terminal R residue is deleted. In someembodiments, the FGF19 variant comprises a subsequence of any of theforegoing sequences. In other embodiments, any combination of two ormore of the foregoing FGF19 variants is also contemplated.

In some embodiments of the methods provided herein, the FGF19 variantconsists of an amino acid sequence set forth in any one of SEQ ID NOS:5-29; or a subsequence or fragment thereof. In certain embodiments, theN-terminal R residue is deleted. In some embodiments, the FGF19 variantconsists of SEQ ID NO:5. In other embodiments, the FGF19 variantconsists of SEQ ID NO:6. In one embodiment, the FGF19 variant consistsof SEQ ID NO:7. In other embodiments, the FGF19 variant consists of SEQID NO:8. In another embodiment, the FGF19 variant consists of SEQ IDNO:9. In some embodiments, the FGF19 variant consists of SEQ ID NO:10.In other embodiments, the FGF19 variant consists of SEQ ID NO:11. Inanother embodiment, the FGF19 variant consists of SEQ ID NO:12. In someembodiments, the FGF19 variant consists of SEQ ID NO:13. In otherembodiments, the FGF19 variant consists of SEQ ID NO:14. In oneembodiment, the FGF19 variant consists of SEQ ID NO:15. In anotherembodiment, the FGF19 variant consists of SEQ ID NO:16. In someembodiments, the FGF19 variant consists of SEQ ID NO:17. In otherembodiments, the FGF19 variant consists of SEQ ID NO:18. In yet otherembodiments, the FGF19 variant consists of SEQ ID NO:19. In someembodiments, the FGF19 variant consists of SEQ ID NO:20. In oneembodiment, the FGF19 variant consists of SEQ ID NO:21. In someembodiments, the FGF19 variant consists of SEQ ID NO:22. In otherembodiments, the FGF19 variant consists of SEQ ID NO:23. In anotherembodiment, the FGF19 variant consists of SEQ ID NO:24. In someembodiments, the FGF19 variant consists of SEQ ID NO:25. In otherembodiments, the FGF19 variant consists of SEQ ID NO:26. In yet otherembodiments, the FGF19 variant consists of SEQ ID NO:27. In someembodiments, the FGF19 variant consists of SEQ ID NO:28. In otherembodiments, the FGF19 variant consists of SEQ ID NO:29. In certainembodiments, the FGF19 variant consists of any one of the foregoingsequences, wherein the N-terminal R residue is deleted. In someembodiments, the FGF19 variant consists of a subsequence of any of theforegoing sequences. In other embodiments, any combination of two ormore of the foregoing FGF19 variants is also contemplated.

As alluded to above, the present disclosure also contemplates variousmodels. Any model can be used that provides reliable, reproducibleresults. The skilled artisan is familiar with models that can be used inconjunction with the subject matter of the present disclosure. In someembodiments, rodent models are used, particularly mouse models. Inaddition to the ob/ob mouse models used in the examples of theExperimental section, db/db, db/ob and DIO models can find use inpracticing aspects of the present disclosure.

One such embodiment is directed to a model for determining whether aFGF19 variant is a candidate for preventing a cancerous disease,disorder or condition in a subject having a metabolic disorder, themodel comprising a subject that (i) does not exhibit an indicia of acancerous condition prior to the administration of an effective amountof FGF19 or FGF19 surrogate, and (ii) exhibits an indicia of a cancerouscondition after the administration of the FGF19 or the FGF19 surrogate;and wherein an indicia of a cancerous condition improves uponadministration of an effective amount of a polypeptide comprising anamino acid sequence set forth in SEQ ID NO:1. In certain embodiments,the polypeptide consists of an amino acid sequence set forth in SEQ IDNO:1.

The present disclosure also contemplates a model for determining whethera FGF19 variant is a candidate for treating a cancerous disease,disorder or condition in a subject having a metabolic disorder, themodel comprising a subject having at least one indicia of cancerresulting from administration of FGF19 or FGF19 surrogate, wherein theindicia of cancer improves upon administration of an effective amount ofa polypeptide comprising an amino acid sequence set forth in SEQ IDNO:1. In certain embodiments, the polypeptide consists of an amino acidsequence set forth in SEQ ID NO:1.

In some embodiments, provided herein is a model for determining whethera FGF19 variant is a candidate for preventing a cancerous disease,disorder or condition in a subject having a metabolic disorder, themodel comprising a subject that i) does not exhibit an indicia of acancerous condition prior to the administration of an effective amountof FGF19 or FGF19 surrogate, and ii) exhibits an indicia of a cancerouscondition after the administration of the FGF19 or FGF19 surrogate; andwherein an indicia of a cancerous condition improves upon administrationof an effective amount of a FGF19 variant.

In other embodiments, provided herein is a model for determining whethera FGF19 variant is a candidate for treating a cancerous disease,disorder or condition in a subject having a metabolic disorder, themodel comprising a subject having at least one indicia of cancerresulting from administration of FGF19 or FGF19 surrogate, wherein theindicia of cancer improves upon administration of an effective amount ofa FGF19 variant.

In certain embodiments of the models provided herein, the FGF19 variantcomprises an amino acid sequence set forth in any one of SEQ IDNOS:5-29; or a subsequence or fragment thereof. In other embodiments ofthe models provided herein, the FGF19 variant consists of an amino acidsequence set forth in any one of SEQ ID NOS: 5-29; or a subsequence orfragment thereof. In certain embodiments, the N-terminal R residue isdeleted. In some embodiments, the FGF19 variant comprises or consists ofSEQ ID NO:5. In other embodiments, the FGF19 variant comprises orconsists of SEQ ID NO:6. In one embodiment, the FGF19 variant comprisesor consists of SEQ ID NO:7. In other embodiments, the FGF19 variantcomprises or consists of SEQ ID NO:8. In another embodiment, the FGF19variant comprises or consists of SEQ ID NO:9. In some embodiments, theFGF19 variant comprises or consists of SEQ ID NO:10. In otherembodiments, the FGF19 variant comprises or consists of SEQ ID NO:11. Inanother embodiment, the FGF19 variant comprises or consists of SEQ IDNO:12. In some embodiments, the FGF19 variant comprises or consists ofSEQ ID NO:13. In other embodiments, the FGF19 variant comprises orconsists of SEQ ID NO:14. In one embodiment, the FGF19 variant comprisesor consists of SEQ ID NO:15. In another embodiment, the FGF19 variantcomprises or consists of SEQ ID NO:16. In some embodiments, the FGF19variant comprises or consists of SEQ ID NO:17. In other embodiments, theFGF19 variant comprises or consists of SEQ ID NO:18. In yet otherembodiments, the FGF19 variant comprises or consists of SEQ ID NO:19. Insome embodiments, the FGF19 variant comprises or consists of SEQ IDNO:20. In one embodiment, the FGF19 variant comprises or consists of SEQID NO:21. In some embodiments, the FGF19 variant comprises or consistsof SEQ ID NO:22. In other embodiments, the FGF19 variant comprises orconsists of SEQ ID NO:23. In another embodiment, the FGF19 variantcomprises or consists of SEQ ID NO:24. In some embodiments, the FGF19variant comprises or consists of SEQ ID NO:25. In other embodiments, theFGF19 variant comprises or consists of SEQ ID NO:26. In yet otherembodiments, the FGF19 variant comprises or consists of SEQ ID NO:27. Insome embodiments, the FGF19 variant comprises or consists of SEQ IDNO:28. In other embodiments, the FGF19 variant comprises or consists ofSEQ ID NO:29. In certain embodiments, the FGF19 variant comprises orconsists of any one of the foregoing sequences, wherein the N-terminal Rresidue is deleted. In some embodiments, the FGF19 variant comprises orconsists of a subsequence of any of the foregoing sequences. In otherembodiments, any combination of two or more of the foregoing FGF19variants is also contemplated.

Evaluation of the Effect of FGF19 Co-Administered with a FGF19 Variant

In the examples set forth in the Experimental section, the impact ondb/db mice of FGF19 administered alone or co-administered with the FGF19variant M70 were assessed. Adeno-associated virus (AAV) was used as thevehicle to deliver and express exogenous genes of interest in the miceand to enable continuous, persistent and systemic exposure to proteinsencoded by the transgenes.

FIG. 2 depicts plasma FGF19 concentrations determined by ELISA in db/dbmice five weeks after AAV-mediated gene delivery of GFP (control); FGF19(two separates doses); and FGF19 and FGF19 variant M70 (two separatedoses of each). The high FGF19 concentrations observed followingco-administration of the FGF19 and M70 transgenes reflect contributionsfrom the expression of both FGF19 variant M70 and FGF19. FGF19-flag wasused in the examples to facilitate quantification of the experimentalresults; as set forth in the Experimental section, the c-Flag componentdid not impact FGF19's tumorogenic effects, though it can have an impactFGF19's antidiabetic effects.

The potential impact of co-administration of FGF19 and FGF19 variant M70on HCC compared to administration of FGF19 alone was evaluated. Asdepicted in FIG. 3, ectopic expression of FGF19 in the db/db mouse modelpromoted the formation of multiple, large, raised tumor nodulesprotruding from the liver surface, whereas livers isolated from miceexpressing both FGF19 and M70 were completely free (under the conditionsemployed) of hepatic nodules. Moreover, FGF19-mediated tumorigenesis, asevidenced by the appearance of hepatic lesions, is completely suppressedwhen the FGF19 and M70 transgenes are co-expressed. These data aresurprising in that they suggest that not only does the engineered FGF19variant M70 lack the tumorigenic potential in mice associated with thewild-type protein, but that it can effectively interfere with theproliferative effects of the wild-type protein. Although a preciseunderstanding of the underlying mechanism of action associated with thisphenomenon is not required in order to practice the present invention,it can be due, at least in part, to competition of M70 for wild-typeFGF19 at the FGF19 binding site.

Example 4 sets forth the effect on mouse body weight initially measuredprior to injection and subsequently determined 3-, 5- and 23-weekspost-injection of the indicated transgenes. As depicted in FIG. 4,transgenic db/db mice co-expressing the FGF19 variant M70 and FGF19showed significant reductions in body weight compared to animals dosedwith control, while less dramatic effects on body weight were observedin mice only expressing the FGF19 transgene. The changes in body weightobserved in mice co-expressing the FGF19 and M70 transgenes were alsoreflected in reduced liver weights compared with weights of liversharvested from animals in the control group.

In a manner similar to that for determination of body weight, the effectof transgene expression on blood glucose was also evaluated prior totransgene injection and 3-, 5- and 23-weeks post-injection. The results,set forth in FIG. 5, indicate that transgenic db/db mice co-expressingthe FGF19 variant M70 and FGF19 show significant reductions in bloodglucose concentrations compared to control animals.

As an expansion of the above studies and date, and as provided inExamples 6-11 of the Experimental section, an in vivo tumorigenicitymodel was established in mice to evaluate FGF19-inducedhepatocarcinogenicity in an effort to target FGF19 in tumorigenesiswithout compromising its essential roles, e.g., in bile acidhomeostasis. Example 7 sets forth an AAV-mediated transgene system forevaluation of hepatocellular tumorigenesis in vivo. FGF19 transgeneexpression was introduced using an AAV-mediated gene delivery approach(Zhang et al., 2009, Hum. Gene Ther. 20, 922-929). As set forth inExample 8, a panel of FGF19 variants was evaluated in vivo andidentified tumor-free variants including M70. Remarkably, as provided inExamples 8 and 9, M70 was shown to retain the beneficial activity ofregulating bile acid homeostasis; and M70 was also shown to bind to andactivate FGFR4, which is assumed to mediate FGF19-associatedtumorigenicity (French et al., 2012, PloS one 7, e36713; Wu et al.,2010, J. Biol. Chem. 285, 5165-5170). As provided in Example 10, FGF19was shown mechanistically to stimulate tumor progression by activatingthe STAT3 pathway, an activity eliminated in M70. Furthermore, asprovided in Example 11, M70 was shown to inhibit FGF19-dependent tumorgrowth in multiple tumor models. Moreover, as provided in Example 12,M70 was shown to inhibit colon tumor growth in a syngenic mouse model.

Thus, in the examples provided herein, natural hormones are engineeredto selectively eliminate potential deleterious activity (i.e.,tumorigenicity), while leaving beneficial function (i.e., bile acidmetabolism) intact. Through extensive structure activity analysis, M70was identified as a tumor-free FGF19 variant that binds and selectiveactivates FGFR4 receptor complex to maintain bile acid homeostasis. Micewith prolonged exposure to supra-physiological levels of M70 (24 weeksin db/db mice, 52 weeks in rasH2 mice) were free of liver tumors(Example 8; FIGS. 7 and 8). M70 was also demonstrated to blockFGF19-associated tumorigenicity in mice and in human cancer xenografts(Example 11, FIG. 11). Although tumor-free FGF19 variants wereidentified previously (Wu et al., 2011, PloS one 6, e17868; Wu et al.,2010a, PNAS, 107, 14158-14163), these variants were specificallydesigned to eliminate FGFR4 binding, and by extension, were impaired inregulating bile acid metabolism. In contrast, M70 exhibits similarpotency and efficacy in binding FGFR4 and regulating Cyp7a1 and pERKpathways downstream of FGFR4 (Examples 9 and 10; FIGS. 9 and 10). Theseresults provide in vivo evidence of selective activation of FGFR4/KLBreceptor complex, which does not lead to hepatocellular tumorigenesis.

The major differences between M70 and FGF19 lie in the N-terminus of theprotein. Each FGF family protein consists of the structurally conservedcentral globular domain, and the flanking N-terminal and C-terminalsegments that are structurally flexible and are divergent in primarysequences (Beenken and Mohammadi, 2009, Nat. Rev. Drug Discov. 8,235-253). In X-ray crystal structures of multiple FGF/FGFR complexes,the N-terminal segment of the FGF molecule makes specific contact withthe FGFR and is believed to play an important role determining thespecificity of the FGF-FGFR interaction (Beenken and Mohammadi, 2009,Nat. Rev. Drug. Discov. 8, 235-253). Through our efforts of a systematicin vivo screen, changing 3 amino acids at the N-terminus coupled with a5-aa deletion was shown to remove tumorigenicity without impairing itsability to activate FGFR4-dependent process such as bile acidregulation.

Without wishing to be bound by theory, several lines of evidenceindicate that M70 exhibits pharmacologic characteristics of a “biasedligand” or a selective modulator. For example, as provided in Example 9and FIG. 9, M70 binds to the extracellular domain of FGFR4 with similarpotency and efficacy as wild type FGF19. M70 activates ERKphosphorylation in cells transfected with FGFR4-KLB or FGFR4 or cellsexpressing FGFR4-KLB endogenously. Like FGF19, M70 potently repressesCyp7a1 in primary hepatocytes and in mice. Unlike FGF19, M70 does notpromote liver tumor formation. Again without wishing to be bound bytheory, the lack of tumorigenicity by M70 could be explained by its lackof activation of pSTAT3, a key signaling molecule in hepatocellularcarcinogenic pathways.

As provided in Example 10 and FIG. 10, FGF19, but not M70, was shown toactivate STAT3 in the liver. STAT3 is a major player in hepatocellularoncogenesis (He and Karin, 2011, Cell Res. 21, 159-168). Phosphorylated(i.e. activated) STAT3 is found in approximate 60% of HCC in human (Heet al., 2010, Cancer Cell 17, 286-297). STAT3 activation also correlateswith poor prognosis in HCC patients (Calvisi et al., 2006,Gastroenterol. 130, 1117-1128). Constitutively-active STAT3 acts as anoncogene in cellular transformation (Bromberg et al., 1999, Cell 98,295-303). Hepatocyte-specific ablation of STAT3 prevented HCCdevelopment in mice (He et al., 2010, Cancer cell 17, 286-297).Inhibitors of STAT3 activation block the growth of human cancer cellsand are being tested in the clinic for treating various cancersincluding HCC (Chen et al., 2010, Clin. Cancer Res, 16, 5189-5199;Karras et al., 2000, Cellular immunol. 202, 124-135; Lin et al., 2009,Oncogene 28, 961-972). IL-6, among other inflammatory cytokines, ispostulated to be the major STAT3 activator in the liver (He et al.,2010, (He et al., 2010, Cancer cell 17, 286-297). IL-6 signaling hasbeen shown to stimulate malignant progression of liver cancerprogenitors (He et al., 2013, Cell 155, 384-396). Increased IL-6production was observed in patients with primary biliary cirrhosis, acholestatic condition associated with increased HCC risk (Kakumu et al.,1993, Gastroenterologia Japonica 28, 18-24). FGF19 is also shown inExample 10 and FIG. 10 to activate STAT3 signaling in vivo. This effectcould be directly mediated by FGFR4 receptor complex, or indirectlythrough induction of cytokines or growth factors. Indeed, the expressionof IL-6 is elevated in the livers of FGF19-treated animals in ourstudies (Example 10; FIG. 10).

M70 may bind to an orthosteric site on FGFR4, since M70 is able todisplace or interfere with FGF19 binding and inhibits FGF19-associatedtumorigenicity. Yet M70 does not block all pathways nondiscriminativelyto the same extent. M70 exhibits bias toward certain FGFR4 signalingpathways (i.e. Cyp7a1, pERK) to the relative exclusion of others (i.e.,tumor), as observed for certain allosteric modulators (FIG. 12).

From a therapeutic perspective, our studies also provide experimentalsupport for the use of M70 in chronic liver diseases and cancer. M70 canbe useful as an anticancer agent for the treatment of FGF19-dependenttumors (see, e.g., Examples, 8 and 11; FIGS. 7, 8 and 11). This isparticularly true given that FGF19 is amplified in ˜15% human HCCs andis upregulated in cirrhosis and cholestatic conditions that often leadto tumor development. While a prior report showed development of aneutralizing anti-FGF19 monoclonal antibody that demonstrated anti-tumoractivity in xenograft models (Desnoyers et al., 2008, Oncogene 27,85-97), such a strategy caused serious adverse effects. Administrationof this antibody to cynomolgus monkeys led to dose-related livertoxicity accompanied by severe diarrhea, due to on-target inhibition ofendogenous FGF19, resulting in increased hepatic bile acid synthesis,elevated serum bile acid, perturbation of enterohepatic circulation, andthe development of diarrhea and liver toxicity ((Pai et al., 2012,Toxicological sciences, 126, 446-456).).

As provided in the Experimental section, and in contrast to neutralizingantibodies, our studies show that M70 retains FGF19's activity on bileacid regulation, while eliminating tumorigenicity (see, e.g., Examples8, 9 and 11; FIGS. 8, 9 and 11). This ensures preservation of bile acidhomeostasis when used as an anti-cancer agent. Importantly, theexperimental data provided herein shows that, not only does M70 lack thetumorigenic potential, but it can also effectively interfere with thetumorigenic effects associated with wild type FGF19. Furthermore, M70inhibits the growth of colon cancer xenograft tumors, in addition toFGF19-mediated HCC. M70 also inhibits the growth of colon cancer in asyngenic mouse model (see, e.g., Example 12 and FIG. 13). These resultsconfirm that, as a selective FGFR4 modulator, M70 antagonizes theoncogenic activity of FGF19.

As provided in the Experimental section, a robust, high throughputsystem was also established to evaluate multiple proteins inhepatocellular tumorigenesis in adult mice using AAV-mediated genedelivery. Overexpression of FGF19 at orthotopic site (liver) was shownto lead to liver dysplasia and the development of HCC in multiplestrains of mice. This eliminates the confounding factors inembryogenesis and development in the traditional transgenic miceapproach. No chemical tumor promoters such as diethylnitrosamine (DEN)or phenobarbital are needed for tumor initiation or promotion. The sameapproach can be adapted to evaluate other oncogenes, signaling proteins,as well as variants of natural proteins.

FGF19 demonstrates an array of biological effects. The therapeuticpotential for FGF19 includes the treatment of chronic liver diseases, aswell as obesity and diabetes, but its promotion of hepatocyteproliferation and carcinogenic potential challenges the development ofFGF19 for chronic use. However, with the identification of M70 as anengineered FGF19 variant devoid of tumorigenicity while retaining itspotent metabolic properties, therapeutic benefits could be achievedwithout unwanted side effects. Our results not only confirm that theselective activation of FGFR4-KLB receptor complex does not induce livertumor formation, but further provide new avenues for utilizing thispathway to treat cancer, diseases with bile acid deregulation, type 2diabetes, and other metabolic disorders.

Polypeptide and Nucleic Acid Molecules

The present disclosure also contemplates active fragments (e.g.,subsequences) of the polypeptides containing contiguous amino acidresidues derived from the polypeptide sequences described herein. Thelength of contiguous amino acid residues of a peptide or a polypeptidesubsequence varies depending on the specific naturally-occurring aminoacid sequence from which the subsequence is derived. In certainembodiments, peptides and polypeptides are from about 5 amino acids toabout 10 amino acids, from about 10 amino acids to about 15 amino acids,from about 15 amino acids to about 20 amino acids, from about 20 aminoacids to about 25 amino acids, from about 25 amino acids to about 30amino acids, from about 30 amino acids to about 40 amino acids, fromabout 40 amino acids to about 50 amino acids, from about 50 amino acidsto about 75 amino acids, from about 75 amino acids to about 100 aminoacids, or from about 100 amino acids up to the full-length polypeptide.

In certain embodiments of the FGF19 variant polypeptides providedherein, the total number of amino acid residues (or mimetics thereof) isless than about 250. In other embodiments, the number of amino acidresidues ranges from about 190 to about 230, from about 200 to about225, or from about 210 to about 220 residues. In still furtherembodiments, the number of amino acid residues is greater than 180,greater than 185, greater than 190, greater than 195, greater than 200,greater than 205, greater than 210, greater than 215, greater than 220or greater than 225 residues.

Additionally, in certain embodiments, the polypeptides have a definedsequence identity compared to a reference sequence over a defined lengthof contiguous amino acids (e.g., a “comparison window”). Methods ofalignment of sequences for comparison are well-known in the art. Optimalalignment of sequences for comparison can be conducted, e.g., by thelocal homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Madison, Wis.), or by manualalignment and visual inspection (see, e.g., Current Protocols inMolecular Biology (Ausubel et al., eds. 1995 supplement)).

As an example, in some embodiments, a suitable polypeptide comprises anamino acid sequence having at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, or at least about 99%, amino acid sequence identity to a contiguousstretch of from about 5 amino acids to about 10 amino acids, from about10 amino acids to about 12 amino acids, from about 12 amino acids toabout 15 amino acids, from about 15 amino acids to about 20 amino acids,from about 20 amino acids to about 25 amino acids, from about 25 aminoacids to about 30 amino acids, from about 30 amino acids to about 35amino acids, from about 35 amino acids to about 40 amino acids, fromabout 40 amino acids to about 45 amino acids, from about 45 amino acidsto about 50 amino acids, from about 50 amino acids to about 60 aminoacids, from about 60 amino acids to about 70 amino acids, from about 70amino acids to about 80 amino acids, from about 80 amino acids to about90 amino acids, from about 90 amino acids to about 100 amino acids, fromabout 100 amino acids to about 110 amino acids, from about 110 aminoacids to about 120 amino acids, from about 120 amino acids to about 130amino acids, from about 130 amino acids to about 140 amino acids, fromabout 140 amino acids to about 150 amino acids, from about 150 aminoacids to about 160 amino acids, from about 160 amino acids to about 170amino acids, from about 170 amino acids to about 180 amino acids, fromabout 180 amino acids to about 190 amino acids, or about 194 aminoacids, of one of the amino acid sequences described herein.

In certain embodiments, the polypeptides are isolated from a naturalsource (e.g., an environment other than its naturally-occurringenvironment) and also can be recombinantly made (e.g., in a geneticallymodified host cell such as bacteria; yeast; Pichia; insect cells; andthe like), where the genetically modified host cell is modified with anucleic acid comprising a nucleotide sequence encoding the polypeptide.The polypeptides can also be synthetically produced (e.g., by cell-freechemical synthesis). Methods of productions are described in more detailbelow.

In some embodiments, a polypeptide is generated using recombinanttechniques to manipulate different FGF19-related nucleic acids known inthe art to provide constructs capable of encoding the polypeptide. Itwill be appreciated that, when provided a particular amino acidsequence, the ordinary skilled artisan will recognize a variety ofdifferent nucleic acid molecules encoding such amino acid sequence inview of her background and experience in, for example, molecularbiology.

In some embodiments, the present disclosure also provides polypeptidesthat have one or more alterations in the amino acid residues (e.g., atlocations that are not conserved across variants or species) compared toa reference sequence (e.g., the corresponding wild-type sequence). Suchpolypeptides frequently retain domains that are conserved among speciesand have the same biological activity as the naturally-occurringpolypeptides. Such polypeptides frequently also have one or moreconservative amino acid substitutions. The phrase “conservative aminoacid substitution” generally refers to substitution of amino acidresidues within the following groups: 1) L, I, M, V, F; 2)R, K; 3) F, Y,H, W, R; 4) G, A, T, S; 5) Q, N; and 6) D, E. Conservative amino acidsubstitutions preserve the activity of the protein by replacing an aminoacid(s) in the protein with an amino acid with a side chain of similaracidity, basicity, charge, polarity, or size of the side chain. Guidancefor substitutions, insertions, or deletions can be based on alignmentsof amino acid sequences of different variant proteins or proteins fromdifferent species.

In particular embodiments, modifications to the Loop-8 region of FGF19are contemplated. Herein, FGF19 residues 127-129 (SEQ ID NO:3) aredefined as constituting the Loop-8 region, although in the literaturethe Loop-8 region is sometimes defined as including or consisting ofother residues (e.g., residues 125-129). Certain combinations of R127Land P128E substitutions to the FGF19 framework had an unexpectedlypositive effect on HCC formation. A combination of R127L and P128Esubstitutions and a substitution of Gln (Q) for Leu (L) in the FGF19core region also had significant effects on preventing HCC formation.

Accordingly, variants of the FGF19 Loop-8 region are included since theycan reduce or eliminate substantial, measurable or detectable HCCformation. Furthermore, the effect of reducing HCC formation may beenhanced by modifications to amino acid residues outside of the Loop-8region (e.g., substitutions of amino acid residues in the core region,such as the region corresponding to amino acids 21-29 of SEQ ID NO:3).

In some embodiments, the Loop-8 modified variant is M70:MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDS16MDPFGLVTGLEAV RSPSFEK(SEQ ID NO:70), comprising a substitution in the FGF19 Loop-8 region(underlined). In certain embodiments, the Loop-8 modified M70 variantcomprises a substitution in the FGF19 Loop-8 region (underlined)corresponding to (i) a R127L substitution, (ii) a P128E substitution, or(iii) a R127L substitution and a P128E substitution. In certainembodiments, the Loop-8 modified M70 variant further comprises orfurther comprises a substitution in the FGF19 core region. In someembodiments, the Loop-8 modified M70 variant comprises a L18Qsubstitution.

In other embodiments, the Loop-8 modified variant is M5 (SEQ ID NO:5),M6 (SEQ ID NO:6), M7 (SEQ ID NO7), M14 (SEQ ID NO:8), M15 (SEQ ID NO:9),M32 (SEQ ID NO:10), M36 (SEQ ID NO:11), M43 (SEQ ID NO:12), M50 (SEQ IDNO:13), M52 (SEQ ID NO14), M53 (SEQ ID NO:15), M67 (SEQ ID NO:16), M68(SEQ ID NO:17), M69 (SEQ ID NO:18), M70 (SEQ ID NO:1 or 19), M75 (SEQ IDNO:20), M76 (SEQ ID NO:21), M77 (SEQ ID NO:22), M83 (SEQ ID NO:23), M84(SEQ ID NO:24), M140 (SEQ ID NO:25), M5-R (SEQ ID NO:26), M6-R (SEQ IDNO:27), M50-R (SEQ ID NO:28), or M160 (SEQ ID NO:29). In someembodiments, the Loop-8 modified variant comprises a substitution in theFGF19 Loop-8 region corresponding to amino acids 127-129 of SEQ ID NO:3.In certain embodiments, the Loop-8 modified variant comprises asubstitution in the FGF19 Loop-8 region corresponding to (i) a R127Lsubstitution, (ii) a P128E substitution, or (iii) a R127L substitutionand a P128E substitution. In some embodiments, the FGF19 variantcomprises or further comprises a substitution in the core regioncorresponding to amino acids 21-29 of SEQ ID NO:3. In certainembodiments, the FGF19 variant comprises or further comprises asubstitution in the core region corresponding to a L22Q substitution.

Nucleic acid molecules encoding the polypeptides described herein arecontemplated by the present disclosure, including theirnaturally-occurring and non-naturally occurring isoforms, allelicvariants and splice variants. The present disclosure also encompassesnucleic acid sequences that vary in one or more bases from anaturally-occurring DNA sequence but still translate into an amino acidsequence that corresponds to a polypeptide due to degeneracy of thegenetic code.

Amide Bond Substitutions

In some cases, a polypeptide includes one or more linkages other thanpeptide bonds, e.g., at least two adjacent amino acids are joined via alinkage other than an amide bond. For example, in order to reduce oreliminate undesired proteolysis or other means of degradation, and/or toincrease serum stability, and/or to restrict or increase conformationalflexibility, one or more amide bonds within the backbone of apolypeptide can be substituted.

In another example, one or more amide linkages (—CO—NH—) in apolypeptide can be replaced with a linkage which is an isostere of anamide linkage, such as —CH₂NH—, CH₂S—, —CH₂CH₂—, —CH═CH-(cis and trans),—COCH₂—, —CH(OH)CH₂— or —CH₂SO—. One or more amide linkages in apolypeptide can also be replaced by, for example, a reduced isosterepseudopeptide bond. See Couder et al. (1993) Int. J. Peptide ProteinRes. 41:181-184. Such replacements and how to effect are known to thoseof ordinary skill in the art.

Amino Acid Substitutions

In certain embodiments, one or more amino acid substitutions are made ina polypeptide. The following are non-limiting examples:

a) a substitution of alkyl-substituted hydrophobic amino acids,including alanine, leucine, isoleucine, valine, norleucine,(S)-2-aminobutyric acid, (S)-cyclohexylalanine or other simplealpha-amino acids substituted by an aliphatic side chain from C₁-C₁₀carbons including branched, cyclic and straight chain alkyl, alkenyl oralkynyl substitutions;

b) a substitution of aromatic-substituted hydrophobic amino acids,including phenylalanine, tryptophan, tyrosine, sulfotyrosine,biphenylalanine, 1-naphthylalanine, 2-naphthylalanine,2-benzothienylalanine, 3-benzothienylalanine, histidine, includingamino, alkylamino, dialkylamino, aza, halogenated (fluoro, chloro,bromo, or iodo) or alkoxy (from C₁-C₄)-substituted forms of theabove-listed aromatic amino acids, illustrative examples of which are:2-, 3- or 4-aminophenylalanine, 2-, 3- or 4-chlorophenylalanine, 2-, 3-or 4-methylphenylalanine, 2-, 3- or 4-methoxyphenylalanine, 5-amino-,5-chloro-, 5-methyl- or 5-methoxytryptophan, 2′-, 3′-, or 4′-amino-,2′-, 3′-, or 4′-chloro-, 2, 3, or 4-biphenylalanine, 2′-, 3′-, or4′-methyl-, 2-, 3- or 4-biphenylalanine, and 2- or 3-pyridylalanine;

c) a substitution of amino acids containing basic side chains, includingarginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid,homoarginine, including alkyl, alkenyl, or aryl-substituted (from C₁-C₁₀branched, linear, or cyclic) derivatives of the previous amino acids,whether the substituent is on the heteroatoms (such as the alphanitrogen, or the distal nitrogen or nitrogens, or on the alpha carbon,in the pro-R position for example. Compounds that serve as illustrativeexamples include: N-epsilon-isopropyl-lysine,3-(4-tetrahydropyridyl)-glycine, 3-(4-tetrahydropyridyl)-alanine,N,N-gamma, gamma′-diethyl-homoarginine. Included also are compounds suchas alpha-methyl-arginine, alpha-methyl-2,3-diaminopropionic acid,alpha-methyl-histidine, alpha-methyl-ornithine where the alkyl groupoccupies the pro-R position of the alpha-carbon. Also included are theamides formed from alkyl, aromatic, heteroaromatic (where theheteroaromatic group has one or more nitrogen, oxygen or sulfur atomssingly or in combination) carboxylic acids or any of the many well-knownactivated derivatives such as acid chlorides, active esters, activeazolides and related derivatives) and lysine, ornithine, or2,3-diaminopropionic acid;

d) substitution of acidic amino acids, including aspartic acid, glutamicacid, homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl, andheteroaryl sulfonamides of 2,4-diaminopriopionic acid, ornithine orlysine and tetrazole-substituted alkyl amino acids;

e) a substitution of side chain amide residue, including asparagine,glutamine, and alkyl or aromatic substitute derivatives of asparagine orglutamine; and/or

f) a substitution of hydroxyl containing amino acids, including serine,threonine, homoserine, 2,3-diaminopropionic acid, and alkyl or aromaticsubstituted derivatives of serine or threonine.

In some embodiments, a polypeptide comprises one or more naturallyoccurring non-genetically encoded L-amino acids, synthetic L-amino acidsor D-enantiomers of an amino acid. For example, a polypeptide cancomprise only D-amino acids. For example, in certain embodiments, apolypeptide comprises one or more of the following residues:hydroxyproline, β-alanine, o-aminobenzoic acid, m-aminobenzoic acid,p-aminobenzoic acid, m-aminomethylbenzoic acid, 2,3-diaminopropionicacid, α-aminoisobutyric acid, N-methylglycine (sarcosine), ornithine,citrulline, t-butylalanine, t-butylglycine, N-methylisoleucine,phenylglycine, cyclohexylalanine, norleucine, naphthylalanine,pyridylalanine 3-benzothienyl alanine, 4-chlorophenylalanine,2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine,penicillamine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid,β-2-thienylalanine, methionine sulfoxide, homoarginine, N-acetyl lysine,2,4-diamino butyric acid, rho-aminophenylalanine, N-methylvaline,homocysteine, homoserine, ε-amino hexanoic acid, ω-aminohexanoic acid,ω-aminoheptanoic acid, ω-aminooctanoic acid, ω-aminodecanoic acid,ω-aminotetradecanoic acid, cyclohexylalanine, α,γ-diaminobutyric acid,α,β-diaminopropionic acid, δ-amino valeric acid, and 2,3-diaminobutyricacid.

Additional Modifications

In some embodiments, a cysteine residue or a cysteine analog isintroduced into a polypeptide to provide for linkage to another peptidevia a disulfide linkage or to provide for cyclization of thepolypeptide. Methods of introducing a cysteine or cysteine analog areknown in the art; see, e.g., U.S. Pat. No. 8,067,532.

In other embodiments, a polypeptide is cyclized. For example, one ormore cysteine or cysteine analogs can be introduced into a polypeptide,where the introduced cysteine or cysteine analog can form a disulfidebond with a second introduced cysteine or cysteine analog. Other meansof cyclization include introduction of an oxime linker or a lanthioninelinker; see, e.g., U.S. Pat. No. 8,044,175. Any combination of aminoacids (or non-amino acid moieties) that can form a cyclizing bond can beused and/or introduced. A cyclizing bond can be generated with anycombination of amino acids (or with amino acid and —(CH₂)_(n)—CO— or—(CH₂)_(n)—C₆H₄—CO—) with functional groups which allow for theintroduction of a bridge. Some examples are disulfides, disulfidemimetics such as the —(CH₂)_(n)— carba bridge, thioacetal, thioetherbridges (cystathionine or lanthionine) and bridges containing esters andethers. In these examples, n can be any integer, but is frequently lessthan ten.

Other exemplary modifications include, for example, an N-alkyl (or aryl)substitution (ψ[CONR]), or backbone crosslinking to construct lactamsand other cyclic structures. Other derivatives of the modulatorcompounds of the present disclosure include C-terminal hydroxymethylderivatives, O-modified derivatives (e.g., C-terminal hydroxymethylbenzyl ether), N-terminally modified derivatives including substitutedamides such as alkylamides and hydrazides.

In some cases, one or more L-amino acids in a polypeptide are replacedwith a D-amino acid.

In some cases, a polypeptide is a retro-inverso analog (Sela and Zisman(1997) FASEB J. 11:449). Retro-inverso peptide analogs are isomers oflinear polypeptides in which the direction of the amino acid sequence isreversed (retro) and the chirality, D- or L-, of one or more amino acidstherein is inverted (inverso) e.g., using D-amino acids rather thanL-amino acids. See, e.g., Jameson et al. (1994) Nature 368:744; andBrady et al. (1994) Nature 368:692.

A polypeptide can include a “Protein Transduction Domain” (PTD), whichrefers to a polypeptide, polynucleotide, carbohydrate, or organic orinorganic compound that facilitates traversing a lipid bilayer, micelle,cell membrane, organelle membrane, or vesicle membrane. A PTD attachedto another molecule facilitates the molecule traversing a membrane, forexample going from extracellular space to intracellular space, orcytosol to within an organelle. In some embodiments, a PTD is covalentlylinked to the amino terminus of a polypeptide, while in otherembodiments, a PTD is covalently linked to the carboxyl terminus of apolypeptide. Exemplary protein transduction domains include, but are notlimited to, a minimal undecapeptide protein transduction domain(corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR;SEQ ID NO:30); a polyarginine sequence comprising a number of argininessufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10,or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer GeneTher. 9(6):489-96); a Drosophila antennapedia protein transductiondomain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); a truncatedhuman calcitonin peptide (Trehin et al. (2004) Pharm. Research21:1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci.USA 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO:31); transportanGWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:32);KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:33); and RQIKIWFQNRRMKWKK(SEQ ID NO:34). Exemplary PTDs include, but are not limited to,YGRKKRRQRRR (SEQ ID NO:30), RKKRRQRRR (SEQ ID NO:35); an argininehomopolymer of from 3 arginine residues to 50 arginine residues.Exemplary PTD domain amino acid sequences include, but are not limitedto, any of the following: YGRKKRRQRRR (SEQ ID NO:30); RKKRRQRR (SEQ IDNO:36); YARAAARQARA (SEQ ID NO:37); THRLPRRRRRR (SEQ ID NO:38); andGGRRARRRRRR (SEQ ID NO:39).

The carboxyl group COR₃ of the amino acid at the C-terminal end of apolypeptide can be present in a free form (R₃═OH) or in the form of aphysiologically-tolerated alkaline or alkaline earth salt such as, e.g.,a sodium, potassium or calcium salt. The carboxyl group can also beesterified with primary, secondary or tertiary alcohols such as, e.g.,methanol, branched or unbranched C₁-C₆-alkyl alcohols, e.g., ethylalcohol or tert-butanol. The carboxyl group can also be amidated withprimary or secondary amines such as ammonia, branched or unbranchedC₁-C₆-alkylamines or C₁-C₆ di-alkylamines, e.g., methylamine ordimethylamine.

The amino group of the amino acid NR₁R₂ at the N-terminus of apolypeptide can be present in a free form (R₁═H and R₂═H) or in the formof a physiologically-tolerated salt such as, e.g., a chloride oracetate. The amino group can also be acetylated with acids such thatR₁=H and R₂=acetyl, trifluoroacetyl, or adamantyl. The amino group canbe present in a form protected by amino-protecting groups conventionallyused in peptide chemistry such as, e.g., Fmoc, Benzyloxycarbonyl (Z),Boc, or Alloc. The amino group can be N-alkylated in which R₁ and/orR₂=C₁-C₆ alkyl or C₂-C₈ alkenyl or C₇-C₉ aralkyl. Alkyl residues can bestraight-chained, branched or cyclic (e.g., ethyl, isopropyl andcyclohexyl, respectively).

Methods of Production of Polypeptides

A polypeptide of the present disclosure can be produced by any suitablemethod, including recombinant and non-recombinant methods (e.g.,chemical synthesis).

A. Chemical Synthesis

Where a polypeptide is chemically synthesized, the synthesis can proceedvia liquid-phase or solid-phase. Solid-phase peptide synthesis (SPPS)allows the incorporation of unnatural amino acids and/or peptide/proteinbackbone modification. Various forms of SPPS, such as Fmoc and Boc, areavailable for synthesizing polypeptides of the present disclosure.Details of the chemical synthesis are known in the art (e.g., Ganesan A.2006 Mini Rev. Med. Chem. 6:3-10; and Camarero J. A. et al, 2005 ProteinPept Lett. 12:723-8).

Solid phase peptide synthesis can be performed as described hereafter.The a functions (Na) and any reactive side chains are protected withacid-labile or base-labile groups. The protective groups are stableunder the conditions for linking amide bonds but can be readily cleavedwithout impairing the peptide chain that has formed. Suitable protectivegroups for the α-amino function include, but are not limited to, thefollowing: t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Z),o-chlorbenzyloxycarbonyl, bi-phenylisopropyloxycarbonyl,tert-amyloxycarbonyl (Amoc), α,α-dimethyl-3,5-dimethoxy-benzyloxycarbonyl, o-nitrosulfenyl,2-cyano-t-butoxy-carbonyl, 9-fluorenylmethoxycarbonyl (Fmoc),1-(4,4-dimethyl-2,6-dioxocylohex-1-ylidene)ethyl (Dde) and the like.

Suitable side chain protective groups include, but are not limited to:acetyl, allyl (All), allyloxycarbonyl (Alloc), benzyl (Bzl),benzyloxycarbonyl (Z), t-butyloxycarbonyl (Boc), benzyloxymethyl (Bom),o-bromobenzyloxycarbonyl, t-butyl (tBu), t-butyldimethylsilyl,2-chlorobenzyl, 2-chlorobenzyloxycarbonyl (2-CIZ), 2,6-dichlorobenzyl,cyclohexyl, cyclopentyl,1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde), isopropyl,4-methoxy-2,3-6-trimethylbenzylsulfonyl (Mtr),2,3,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), pivalyl,tetrahydropyran-2-yl, tosyl (Tos), 2,4,6-trimethoxybenzyl,trimethylsilyl and trityl (Trt).

In the solid phase synthesis, the C-terminal amino acid is coupled to asuitable support material. Suitable support materials are those whichare inert towards the reagents and reaction conditions for the step-wisecondensation and cleavage reactions of the synthesis process and whichdo not dissolve in the reaction media being used. Examples ofcommercially-available support materials include styrene/divinylbenzenecopolymers which have been modified with reactive groups and/orpolyethylene glycol; chloromethylated styrene/divinylbenzene copolymers;hydroxymethylated or aminomethylated styrene/divinylbenzene copolymersand the like. Polystyrene (1%)-divinylbenzene or TentaGel® derivatizedwith 4-benzyloxybenzyl-alcohol (Wang-anchor) or 2-chlorotrityl chloridecan be used if it is intended to prepare the peptidic acid. In the caseof the peptide amide, polystyrene (1%) divinylbenzene or TentaGel®derivatized with 5-(4′-aminomethyl)-3′,5′-dimethoxyphenoxy)valeric acid(PAL-anchor) or p-(2,4-dimethoxyphenyl-amino methyl)-phenoxy group (Rinkamide anchor) can be used.

The linkage to the polymeric support can be achieved by reacting theC-terminal Fmoc-protected amino acid with the support material with theaddition of an activation reagent in ethanol, acetonitrile,N,N-dimethylformamide (DMF), dichloromethane, tetrahydrofuran,N-methylpyrrolidone or similar solvents at room temperature or elevatedtemperatures (e.g., between 40° C. and 60° C.) and with reaction timesof, e.g., 2 to 72 hours.

The coupling of the Nα-protected amino acid (e.g., the Fmoc amino acid)to the PAL, Wang or Rink anchor can, for example, be carried out withthe aid of coupling reagents such as N,N′-dicyclohexylcarbodiimide(DCC), N,N′-diisopropylcarbodiimide (DIC) or other carbodiimides,2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU) or other uronium salts, o-acyl-ureas,benzotriazol-1-yl-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP) or other phosphonium salts, N-hydroxysuccinimides, otherN-hydroxyimides or oximes in the presence or also in the absence of1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole, e.g., with theaid of TBTU with addition of HOBt, with or without the addition of abase such as, for example, diisopropylethylamine (DIEA), triethylamineor N-methylmorpholine, e.g., diisopropylethylamine with reaction timesof 2 to 72 hours (e.g., 3 hours in a 1.5 to 3-fold excess of the aminoacid and the coupling reagents, e.g., in a 2-fold excess and attemperatures between about 10° C. and 50° C., e.g., 25° C. in a solventsuch as dimethylformamide, N-methylpyrrolidone or dichloromethane, e.g.,dimethylformamide).

Instead of the coupling reagents, it is also possible to use the activeesters (e.g., pentafluorophenyl, p-nitrophenyl or the like), thesymmetric anhydride of the Nα-Fmoc-amino acid, its acid chloride or acidfluoride under the conditions described above.

The Nα-protected amino acid (e.g., the Fmoc amino acid) can be coupledto the 2-chlorotrityl resin in dichloromethane with the addition of DIEAwith reaction times of 10 to 120 minutes, e.g., 20 minutes, but is notlimited to the use of this solvent and this base.

The successive coupling of the protected amino acids can be carried outaccording to conventional methods in peptide synthesis, typically in anautomated peptide synthesizer. After cleavage of the Nα-Fmoc protectivegroup of the coupled amino acid on the solid phase by treatment with,e.g., piperidine (10% to 50%) in dimethylformamide for 5 to 20 minutes,e.g., 2×2 minutes with 50% piperidine in DMF and 1×15 minutes with 20%piperidine in DMF, the next protected amino acid in a 3 to 10-foldexcess, e.g., in a 10-fold excess, is coupled to the previous amino acidin an inert, non-aqueous, polar solvent such as dichloromethane, DMF ormixtures of the two and at temperatures between about 10° C. and 50° C.,e.g., at 25° C. The previously mentioned reagents for coupling the firstNα-Fmoc amino acid to the PAL, Wang or Rink anchor are suitable ascoupling reagents. Active esters of the protected amino acid, orchlorides or fluorides or symmetric anhydrides thereof can also be usedas an alternative.

At the end of the solid phase synthesis, the peptide is cleaved from thesupport material while simultaneously cleaving the side chain protectinggroups. Cleavage can be carried out with trifluoroacetic acid or otherstrongly acidic media with addition of 5%-20% V/V of scavengers such asdimethylsulfide, ethylmethylsulfide, thioanisole, thiocresol, m-cresol,anisole ethanedithiol, phenol or water, e.g., 15% v/vdimethylsulfide/ethanedithiol/m-cresol 1:1:1, within 0.5 to 3 hours,e.g., 2 hours. Peptides with fully protected side chains are obtained bycleaving the 2-chlorotrityl anchor with glacial aceticacid/trifluoroethanol/dichloromethane 2:2:6. The protected peptide canbe purified by chromatography on silica gel. If the peptide is linked tothe solid phase via the Wang anchor and if it is intended to obtain apeptide with a C-terminal alkylamidation, the cleavage can be carriedout by aminolysis with an alkylamine or fluoroalkylamine. The aminolysisis carried out at temperatures between about −10° C. and 50° C. (e.g.,about 25° C.), and reaction times between about 12 and 24 hours (e.g.,about 18 hours). In addition the peptide can be cleaved from the supportby re-esterification, e.g., with methanol.

The acidic solution that is obtained can be admixed with a 3 to 20-foldamount of cold ether or n-hexane, e.g., a 10-fold excess of diethylether, in order to precipitate the peptide and hence to separate thescavengers and cleaved protective groups that remain in the ether. Afurther purification can be carried out by re-precipitating the peptideseveral times from glacial acetic acid. The precipitate that is obtainedcan be taken up in water or tert-butanol or mixtures of the twosolvents, e.g., a 1:1 mixture of tert-butanol/water, and freeze-dried.

The peptide obtained can be purified by various chromatographic methods,including ion exchange over a weakly basic resin in the acetate form;hydrophobic adsorption chromatography on non-derivatizedpolystyrene/divinylbenzene copolymers (e.g., Amberlite® XAD); adsorptionchromatography on silica gel; ion exchange chromatography, e.g., oncarboxymethyl cellulose; distribution chromatography, e.g., on Sephadex®G-25; countercurrent distribution chromatography; or high pressureliquid chromatography (HPLC) e.g., reversed-phase HPLC on octyl oroctadecylsilylsilica (ODS) phases.

B. Recombinant Production

Where a polypeptide is produced using recombinant techniques, thepolypeptide can be produced as an intracellular protein or as a secretedprotein, using any suitable construct and any suitable host cell, whichcan be a prokaryotic or eukaryotic cell, such as a bacterial (e.g., E.coli) or a yeast host cell, respectively. Other examples of eukaryoticcells that can be used as host cells include insect cells, mammaliancells, and/or plant cells. Where mammalian host cells are used, they caninclude human cells (e.g., HeLa, 293, H9 and Jurkat cells); mouse cells(e.g., NIH3T3, L cells, and C127 cells); primate cells (e.g., Cos 1, Cos7 and CV1) and hamster cells (e.g., Chinese hamster ovary (CHO) cells).

A variety of host-vector systems suitable for the expression of apolypeptide can be employed according to standard procedures known inthe art. See, e.g., Sambrook et al., 1989 Current Protocols in MolecularBiology Cold Spring Harbor Press, New York; and Ausubel et al. 1995Current Protocols in Molecular Biology, Eds. Wiley and Sons. Methods forintroduction of genetic material into host cells include, for example,transformation, electroporation, conjugation, calcium phosphate methodsand the like. The method for transfer can be selected so as to providefor stable expression of the introduced polypeptide-encoding nucleicacid. The polypeptide-encoding nucleic acid can be provided as aninheritable episomal element (e.g., a plasmid) or can be genomicallyintegrated. A variety of appropriate vectors for use in production of apolypeptide of interest are commercially available.

Vectors can provide for extrachromosomal maintenance in a host cell orcan provide for integration into the host cell genome. The expressionvector provides transcriptional and translational regulatory sequences,and can provide for inducible or constitutive expression where thecoding region is operably-linked under the transcriptional control ofthe transcriptional initiation region, and a transcriptional andtranslational termination region. In general, the transcriptional andtranslational regulatory sequences can include, but are not limited to,promoter sequences, ribosomal binding sites, transcriptional start andstop sequences, translational start and stop sequences, and enhancer oractivator sequences. Promoters can be either constitutive or inducible,and can be a strong constitutive promoter (e.g., T7).

Expression constructs generally have convenient restriction siteslocated near the promoter sequence to provide for the insertion ofnucleic acid sequences encoding proteins of interest. A selectablemarker operative in the expression host can be present to facilitateselection of cells containing the vector. Moreover, the expressionconstruct can include additional elements. For example, the expressionvector can have one or two replication systems, thus allowing it to bemaintained in organisms, for example, in mammalian or insect cells forexpression and in a prokaryotic host for cloning and amplification. Inaddition, the expression construct can contain a selectable marker geneto allow the selection of transformed host cells. Selectable genes arewell known in the art and will vary with the host cell used.

Isolation and purification of a protein can be accomplished according tomethods known in the art. For example, a protein can be isolated from alysate of cells genetically modified to express the proteinconstitutively and/or upon induction, or from a synthetic reactionmixture by immunoaffinity purification, which generally involvescontacting the sample with an anti-protein antibody, washing to removenon-specifically bound material, and eluting the specifically boundprotein. The isolated protein can be further purified by dialysis andother methods normally employed in protein purification methods. In oneembodiment, the protein can be isolated using metal chelatechromatography methods. Proteins can contain modifications to facilitateisolation.

The polypeptides can be prepared in substantially pure or isolated form(e.g., free from other polypeptides). The polypeptides can be present ina composition that is enriched for the polypeptide relative to othercomponents that can be present (e.g., other polypeptides or other hostcell components). For example, purified polypeptide can be provided suchthat the polypeptide is present in a composition that is substantiallyfree of other expressed proteins, e.g., less than 90%, less than 60%,less than 50%, less than 40%, less than 30%, less than 20%, less than10%, less than 5%, or less than 1%, of the composition is made up ofother expressed proteins.

Therapeutic and Prophylactic Uses

Also provided herein are methods for treating or preventinghyperglycemia, hyperinsulinemia, glucose intolerance, glucose metabolismdisorders, obesity and other body weight disorders, as well as othermetabolic and metabolic-associated diseases, disorders and conditions bythe administration of the agents, or compositions thereof. Furthermore,as described herein, the present disclosure provides methods fortreating or preventing a host of other diseases, disorders andconditions. Such methods can also have an advantageous effect on one ormore symptoms associated with a disease, disorder or condition by, forexample, decreasing the severity or the frequency of a symptom. Incertain embodiments, the method is a method for treating a disease ordisorder. In other embodiments, the method is a method for preventing adisease or disorder.

In certain embodiments, the present disclosure contemplates methods oftreating (or preventing, in certain circumstances) a subject having ametabolic disorder, the method comprising providing a subject having ametabolic disorder, wherein the subject exhibits an indicia of aFGF19-induced cancerous condition, and administering to the subject atherapeutically effective amount of a FGF19 variant identified from apool of candidate FGF19 variant polypeptides as described herein;wherein there is an improvement in the metabolic disorder in thesubject.

Non-limiting examples of diseases, disorders and conditions include: 1)glucose utilization disorders and the sequelae associated therewith,including diabetes mellitus (type I and type-2), gestational diabetes,hyperglycemia, insulin resistance, abnormal glucose metabolism,“pre-diabetes” (Impaired Fasting Glucose (IFG) or Impaired GlucoseTolerance (IGT)), and other physiological disorders associated with, orthat result from, the hyperglycemic condition, including, for example,histopathological changes such as pancreatic β-cell destruction. Furtherhyperglycemic-related disorders include kidney damage (e.g., tubuledamage or nephropathy), liver degeneration, eye damage (e.g., diabeticretinopathy or cataracts), and diabetic foot disorders; 2) dyslipidemiasand their sequelae such as, for example, atherosclerosis, coronaryartery disease, cerebrovascular disorders and the like; 3) otherconditions which can be associated with the metabolic syndrome, such asobesity and elevated body mass (including the co-morbid conditionsthereof such as, but not limited to, nonalcoholic fatty liver disease(NAFLD), nonalcoholic steatohepatitis (NASH), and polycystic ovariansyndrome (PCOS)), and also include thromboses, hypercoagulable andprothrombotic states (arterial and venous), hypertension, cardiovasculardisease, stroke and heart failure; 4) disorders or conditions in whichinflammatory reactions are involved, including atherosclerosis, chronicinflammatory bowel diseases (e.g., Crohn's disease and ulcerativecolitis), asthma, lupus erythematosus, arthritis, or other inflammatoryrheumatic disorders; 5) disorders of cell cycle or cell differentiationprocesses such as adipose cell tumors, lipomatous carcinomas including,for example, liposarcomas, solid tumors, and neoplasms; 6)neurodegenerative diseases and/or demyelinating disorders of the centraland peripheral nervous systems and/or neurological diseases involvingneuroinflammatory processes and/or other peripheral neuropathies,including Alzheimer's disease, multiple sclerosis, Parkinson's disease,progressive multifocal leukoencephalopathy and Guillian-Barre syndrome;7) skin and dermatological disorders and/or disorders of wound healingprocesses, including erythemato-squamous dermatoses; and 8) otherdisorders such as syndrome X, osteoarthritis, and acute respiratorydistress syndrome.

In order to determine whether a subject can be a candidate for thetreatment or prevention of hyperglycemia, hyperinsulinemia, glucoseintolerance, and/or glucose disorders by the methods provided herein,various diagnostic methods known in the art can be utilized (e.g.,fasting plasma glucose (FPG) evaluation and the oral glucose tolerancetest (oGTT)).

In order to determine whether a subject can be a candidate for thetreatment or prevention of a body weight disorder (e.g., obesity) by themethods provided herein, parameters such as, but not limited to, theetiology and the extent of the subject's condition (e.g., how overweightthe subject is compared to reference healthy individual) should beevaluated. For example, an adult having a BMI between ˜25 and ˜29.9kg/m² can be considered overweight (pre-obese), while an adult having aBMI of ˜30 kg/m² or higher can be considered obese. For subjects who areoverweight and/or who have poor diets (e.g., diets high in fat andcalories), it is common to initially implement and assess the effect ofmodified dietary habits and/or exercise regimens before initiating acourse of therapy comprising one or more of the polypeptides of thepresent disclosure.

Also provided herein is a method of treating a subject (e.g., an animal,such as a human) having a metabolic or metabolic-associated disease,disorder or condition, said method comprising: (i) determining whether atest subject having a metabolic disorder is a candidate for treatmentwith a FGF19 variant, the method comprising: (a) providing a testsubject having an indicia of a cancerous condition, the subject having ametabolic disorder, (b) co-administering FGF19 or a FGF19 surrogate, anda FGF19 variant to the test subject, wherein the amount of the FGF19 orthe FGF19 surrogate administered to the test subject is sufficient toinduce a cancerous condition in a reference population, and (c)determining whether an indicia of a cancerous condition is enhanced inthe test subject; wherein the absence of enhancement of an indicia of acancerous condition indicates that the test subject is a candidate fortreatment with a FGF19 variant; and wherein if there is an absence ofenhancement of the indicia of a cancerous condition in the test subject,then the method further comprises (ii) subsequently administering theFGF19 variant to the subject (e.g., an animal, such as a human). Incertain embodiments, the subsequent administration of the FGF19 variantis a therapeutically effective amount resulting in the treatment of themetabolic or metabolic-associated disease, disorder or condition in thesubject (e.g., an animal, such as a human).

Also provided herein is a method of treating a subject (e.g., an animal,such as a human) having a metabolic or metabolic-associated disease,disorder or condition, said method comprising: (i) determining whether atest subject having a metabolic disorder is a candidate for treatmentwith a FGF19 variant, the method comprising: (a) providing a testsubject having an indicia of a cancerous condition, the test subjecthaving a metabolic disorder, and (b) co-administering FGF19 or a FGF19surrogate, and a FGF19 variant to the test subject, wherein the amountof the FGF19 or the FGF19 surrogate is administered to the test subjectis sufficient to induce a cancerous condition in a reference population,and (c) determining whether an indicia of a cancerous condition isreduced in the test subject; wherein the reduction of an indicia of acancerous condition indicates that the test subject is a candidate fortreatment with a FGF19 variant; and wherein if there is a reduction ofthe indicia of a cancerous condition in the test subject, then themethod further comprises (ii) subsequently administering the FGF19variant to the subject (e.g., an animal, such as a human). In certainembodiments, the subsequent administration of the FGF19 variant is atherapeutically effective amount resulting in the treatment of themetabolic or metabolic-associated disease, disorder or condition in thesubject (e.g., an animal, such as a human).

Also provided herein is a method of treating a subject (e.g., an animal,such as a human) having a metabolic or metabolic-associated disease,disorder or condition, said method comprising: (i) determining whether aFGF19 variant is a candidate for treating a test subject having ametabolic disorder, the method comprising: (a) co-administering FGF19 ora FGF19 surrogate, and the FGF19 variant to the test subject having ametabolic disorder, wherein the amount of the FGF19 or the FGF19surrogate administered to the test subject is sufficient to induce acancerous condition in a reference population, and (b) determiningwhether an indicia of a cancerous condition is observed in the testsubject; wherein the absence of an indicia of a cancerous conditionindicates that the FGF19 variant is a candidate for treatment of thetest subject; and wherein if there is an absence of an indicia of acancerous condition, then the method further comprises (ii) subsequentlyadministering the FGF19 variant to the subject (e.g., an animal, such asa human). In certain embodiments, the subsequent administration of theFGF19 variant is a therapeutically effective amount resulting in thetreatment of the metabolic or metabolic-associated disease, disorder orcondition in the subject (e.g., an animal, such as a human).

Also provided herein is a method of treating a subject (e.g., an animal,such as a human) having a metabolic or metabolic-associated disease,disorder or condition, said method comprising: (i) determining whether aFGF19 variant is a candidate for treating a test subject having ametabolic disorder, the method comprising: (a) providing a test subjecthaving a metabolic disorder, the test subject having an indicia of acancerous condition, (b) co-administering FGF19 or a FGF19 surrogate,and a FGF19 variant to the test subject, wherein the amount of the FGF19or the FGF19 surrogate is administered to the test subject is sufficientto exacerbate a cancerous condition in a reference population, and (c)determining whether an indicia of a cancerous condition is enhanced inthe test subject; wherein the absence of exacerbation of an indicia of acancerous condition indicates that the FGF19 variant is a candidate fortreatment of the test subject; and wherein if there is an absence ofenhancement of the indicia of a cancerous condition in the test subject,then the method further comprises (ii) subsequently administering theFGF19 variant to the subject (e.g., an animal, such as a human). Incertain embodiments, the subsequent administration of the FGF19 variantis a therapeutically effective amount resulting in the treatment of themetabolic or metabolic-associated disease, disorder or condition in thesubject (e.g., an animal, such as a human). In some embodiments, one ormore indicia of a cancerous condition are reduced in the test subject.

Also provided herein is a method of treating a subject having ametabolic or metabolic-associated disease, disorder or condition,comprising: (a) providing a subject having a metabolic disorder, whereinthe subject exhibits an indicia of a FGF19-induced cancerous condition,and (b) administering to the subject a therapeutically effective amountof a FGF19 variant identified in any one of methods or models providedherein; wherein there is an improvement in the metabolic ormetabolic-associated disease, disorder or condition in the subject.

In certain embodiments of the methods provided herein, the subject is ananimal. In other embodiments, the subject is a human. In someembodiments, the cancerous condition is a tumor. In certain embodiments,the tumor is a colon tumor or a hepatic tumor. In some embodiments, themetabolic or metabolic-associated disease, disorder or condition is ametabolic disorder. In some embodiments, the metabolic disorder isselected from the group consisting of a hyperglycemic condition, insulinresistance, hyperinsulinemia, glucose intolerance, obesity and metabolicsyndrome. In one embodiment, the hyperglycemic condition is diabetes. Inanother embodiment, the treatment results in an improvement in themetabolic disorder. In certain embodiments, the improvement of themetabolic disorder is a decrease in blood glucose. In other embodiments,the improvement in the metabolic disorder in the subject is a decreasein body weight. In certain embodiments, the improvement in the metabolicdisorder in the subject is a decrease in insulin.

In another aspect, provided herein is a method of antagonizing theoncogenic activity of FGF19, for example, using a FGF19 variant providedherein. In some embodiments, a cell expressing FGF19 is contacted with aFGF19 variant provided herein. In some embodiments, the FGF19 variant isM70. In certain embodiments, provided herein is a method of antagonizingthe oncogenic activity of FGF19 in a subject, comprising administeringto the subject a therapeutically effective amount of a FGF19 variant,thereby antagonizing the oncogenic activity of FGF19 in the subject. Insome embodiments, provided is a method of preventing a FGF19-dependentdisease, disorder or condition, or a symptom thereof, in a subject,comprising administering to the subject a therapeutically effectiveamount of a FGF19 variant, wherein the disease, disorder, condition, orsymptom thereof is prevented in the subject. In other embodiments,provided is a method of treating a FGF19-dependent disease, disorder orcondition, or a symptom thereof, in a subject, comprising administeringto the subject a therapeutically effective amount of a FGF19 variant,wherein the disease, disorder, condition, or symptom thereof is treatedin the subject.

In certain embodiments, the subject has a metabolic disorder and/or anindicia of a cancerous condition. In certain embodiments, theFGF19-dependent disease, disorder or condition is a cancer or tumor. Insome embodiments, the cancer or tumor is a liver, colon, prostate orlung cancer or tumor. In some embodiments, the cancer or tumor isbenign. In other embodiments, the cancer or tumor is malignant.

In certain embodiments, the subject has or is at risk of developing aFGF19-dependent disease, disorder or condition. In some embodiments, theFGF19-dependent disease, disorder or condition is a liver(hepatocellular) disease, disorder or condition, such as cirrhosis orcholestasis. In some embodiments, the liver disease or disorder is achronic liver disease or disorder. In some embodiments, theFGF19-dependent disease, disorder or condition is cancer or tumor, suchas HCC. In other embodiments, the FGF19-dependent disease, disorder orcondition is not a liver disease, disorder or condition, such ascirrhosis or cholestasis. In some embodiments, the FGF19-dependentdisease, disorder or condition is not a cancer or tumor, such as HCC. Insome embodiments, the FGF19-dependent disease, disorder or condition isa colon cancer or tumor. In certain embodiments, the colon cancer ortumor is a colon adenocarcinoma. In some embodiments, theFGF19-dependent disease, disorder or condition is a prostate cancer ortumor. In yet other embodiments, the FGF19-dependent disease, disorderor condition is a lung cancer or tumor. In certain embodiments, the lungcancer or tumor is a lung squamous cell carcinoma. In some embodiments,FGF19 is expressed in a primary or metastatic cancer or tumor cell. Incertain embodiments, the FGF19-dependent disease, disorder or conditionis pre-cancerous. For example, cirrhosis and cholestasis sometimes tolead to liver cancers, such as HCC, and methods of treating orpreventing such liver diseases and disorders are contemplated. Incertain embodiments, the subject is a subject in need of prevention ortreatment thereof. In some embodiments, administration of the FGF19variant maintains bile acid homeostasis in the subject.

Also provided herein is a method of treating a cancer or tumor, such asa FGF19-dependent cancer or tumor, or a symptom thereof, in a subject,comprising administering to the subject a therapeutically effectiveamount of a FGF19 variant. In certain embodiments, the administrationresults in an improvement in the cancer, tumor or symptom thereof in thesubject. In some embodiments, the method results in a reduction in tumornumber, tumor size, or tumor weight. Also provided herein is a method ofpreventing a cancer or tumor, such as a FGF19-dependent cancer or tumor,or a symptom thereof, in a subject, comprising administering to thesubject a therapeutically effective amount of a FGF19 variant. In someembodiments, the administration results in prevention of the cancer,tumor, or symptom thereof in the subject. In some embodiments, themethod results in a reduction in tumor number, tumor size, or tumorweight. In a specific embodiment, the cancer or tumor is aFGF19-dependent cancer or tumor. In certain embodiments, the cancer ortumor is hepatocellular carcinoma. In some embodiments, the cancer ortumor is not hepatocellular carcinoma. In one embodiment, the cancer ortumor is a colon cancer or tumor. In some embodiments, the cancer ortumor is a prostate cancer or tumor. In certain embodiments, the canceror tumor is a lung cancer or tumor. In certain embodiments, the FGF19variant is a polypeptide comprising an amino acid sequence set forth inSEQ ID NO:1. In some embodiments, the FGF19 variant is a polypeptideconsisting of an amino acid sequence set forth in SEQ ID NO:1. Incertain embodiments, the subject is a subject in need thereof.

It is understood that any of the therapeutic or prophylactic methodsprovided herein can be used in conjunction with any of the models orother methods provided herein.

In certain embodiments of the methods provided herein, the FGF19 variantis selected from the group consisting of M5, M6, M7, M14, M15, M32, M36,M43, M52, M53, M67, M68, M69, M70, M75, M76, M77, M83, M84, M140, M144,M145, M146 and M160. In one embodiment, the FGF19 variant is M5. Inanother embodiment, the FGF19 variant is M6. In some embodiments, theFGF19 variant is M7. In one embodiment, the FGF19 variant is M14. Inanother embodiment, the FGF19 variant is M15. In other embodiments, theFGF19 variant is M32. In one embodiment, the FGF19 variant is M36. Inanother embodiment, the FGF19 variant is M43. In other embodiments, theFGF19 variant is M52. In yet other embodiment, the FGF19 variant is M53.In some embodiments, the FGF19 variant is M67. In one embodiment, theFGF19 variant is M68. In another embodiment, the FGF19 variant is M69.In some embodiments, the FGF19 variant is M70. In one embodiment, theFGF19 variant is M75. In another embodiment, the FGF19 variant is M76.In other embodiments, the FGF19 variant is M77. In yet otherembodiments, the FGF19 variant is M83. In one embodiment, the FGF19variant is M84. In another embodiment, the FGF19 variant is M140. Inother embodiments, the FGF19 variant is M144. In yet other embodiments,the FGF19 variant is M145. In one embodiment, the FGF19 variant is M146.In some embodiments, the FGF19 variant is M160. In other embodiments,any combination of two or more of the foregoing FGF19 variants is alsocontemplated.

In some embodiments of the methods provided herein, the FGF19 variantcomprises an amino acid sequence set forth in any one of SEQ IDNOS:5-29; or a subsequence or fragment thereof. In other embodiments ofthe methods provided herein, the FGF19 variant consists of an amino acidsequence set forth in any one of SEQ ID NOS: 5-29; or a subsequence orfragment thereof. In certain embodiments, the N-terminal R residue isdeleted. In some embodiments, the FGF19 variant comprises or consists ofSEQ ID NO:5. In other embodiments, the FGF19 variant comprises orconsists of SEQ ID NO:6. In one embodiment, the FGF19 variant comprisesor consists of SEQ ID NO:7. In other embodiments, the FGF19 variantcomprises or consists of SEQ ID NO:8. In another embodiment, the FGF19variant comprises or consists of SEQ ID NO:9. In some embodiments, theFGF19 variant comprises or consists of SEQ ID NO:10. In otherembodiments, the FGF19 variant comprises or consists of SEQ ID NO:11. Inanother embodiment, the FGF19 variant comprises or consists of SEQ IDNO:12. In some embodiments, the FGF19 variant comprises or consists ofSEQ ID NO:13. In other embodiments, the FGF19 variant comprises orconsists of SEQ ID NO:14. In one embodiment, the FGF19 variant comprisesor consists of SEQ ID NO:15. In another embodiment, the FGF19 variantcomprises or consists of SEQ ID NO:16. In some embodiments, the FGF19variant comprises or consists of SEQ ID NO:17. In other embodiments, theFGF19 variant comprises or consists of SEQ ID NO:18. In yet otherembodiments, the FGF19 variant comprises or consists of SEQ ID NO:19. Insome embodiments, the FGF19 variant comprises or consists of SEQ IDNO:20. In one embodiment, the FGF19 variant comprises or consists of SEQID NO:21. In some embodiments, the FGF19 variant comprises or consistsof SEQ ID NO:22. In other embodiments, the FGF19 variant comprises orconsists of SEQ ID NO:23. In another embodiment, the FGF19 variantcomprises or consists of SEQ ID NO:24. In some embodiments, the FGF19variant comprises or consists of SEQ ID NO:25. In other embodiments, theFGF19 variant comprises or consists of SEQ ID NO:26. In yet otherembodiments, the FGF19 variant comprises or consists of SEQ ID NO:27. Insome embodiments, the FGF19 variant comprises or consists of SEQ IDNO:28. In other embodiments, the FGF19 variant comprises or consists ofSEQ ID NO:29. In certain embodiments, the FGF19 variant comprises orconsists of any one of the foregoing sequences, wherein the N-terminal Rresidue is deleted. In some embodiments, the FGF19 variant comprises orconsists of a subsequence of any of the foregoing sequences. In otherembodiments, any combination of two or more of the foregoing FGF19variants is also contemplated.

Pharmaceutical Compositions

The polypeptides of the present disclosure can be in the form ofcompositions suitable for administration to a subject. In general, suchcompositions are “pharmaceutical compositions” comprising one or morepolypeptides and one or more pharmaceutically acceptable orphysiologically acceptable diluents, carriers or excipients. In certainembodiments, the polypeptides are present in a therapeuticallyacceptable amount. The pharmaceutical compositions can be used in themethods of the present disclosure; thus, for example, the pharmaceuticalcompositions can be administered ex vivo or in vivo to a subject inorder to practice the therapeutic and prophylactic methods and usesdescribed herein.

The pharmaceutical compositions of the present disclosure can beformulated to be compatible with the intended method or route ofadministration; exemplary routes of administration are set forth herein.Furthermore, the pharmaceutical compositions can be used in combinationwith other therapeutically active agents or compounds (e.g., glucoselowering agents) as described herein in order to treat or prevent thediseases, disorders and conditions as contemplated by the presentdisclosure.

The pharmaceutical compositions typically comprise a therapeuticallyeffective amount of at least one of the polypeptides contemplated by thepresent disclosure and one or more pharmaceutically and physiologicallyacceptable formulation agents. Suitable pharmaceutically acceptable orphysiologically acceptable diluents, carriers or excipients include, butare not limited to, antioxidants (e.g., ascorbic acid and sodiumbisulfate), preservatives (e.g., benzyl alcohol, methyl parabens, ethylor n-propyl, p-hydroxybenzoate), emulsifying agents, suspending agents,dispersing agents, solvents, fillers, bulking agents, detergents,buffers, vehicles, diluents, and/or adjuvants. For example, a suitablevehicle can be physiological saline solution or citrate buffered saline,possibly supplemented with other materials common in pharmaceuticalcompositions for parenteral administration. Neutral buffered saline orsaline mixed with serum albumin are further exemplary vehicles. Thoseskilled in the art will readily recognize a variety of buffers thatcould be used in the pharmaceutical compositions and dosage forms.Typical buffers include, but are not limited to, pharmaceuticallyacceptable weak acids, weak bases, or mixtures thereof. As an example,the buffer components can be water soluble materials such as phosphoricacid, tartaric acids, lactic acid, succinic acid, citric acid, aceticacid, ascorbic acid, aspartic acid, glutamic acid, and salts thereof.Acceptable buffering agents include, for example, a Tris buffer,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS), andN-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS).

After a pharmaceutical composition has been formulated, it can be storedin sterile vials as a solution, suspension, gel, emulsion, solid, ordehydrated or lyophilized powder. Such formulations can be stored eitherin a ready-to-use form, a lyophilized form requiring reconstitutionprior to use, a liquid form requiring dilution prior to use, or otheracceptable form. In some embodiments, the pharmaceutical composition isprovided in a single-use container (e.g., a single-use vial, ampoule,syringe, or autoinjector (similar to, e.g., an EpiPen®)), whereas amulti-use container (e.g., a multi-use vial) is provided in otherembodiments. Any drug delivery apparatus can be used to deliver thepolypeptides, including implants (e.g., implantable pumps) and cathetersystems, both of which are well known to the skilled artisan. Depotinjections, which are generally administered subcutaneously orintramuscularly, can also be utilized to release the polypeptidesdisclosed herein over a defined period of time. Depot injections areusually either solid- or oil-based and generally comprise at least oneof the formulation components set forth herein. One of ordinary skill inthe art is familiar with possible formulations and uses of depotinjections. In certain embodiments, the use of Nano Precision Medical'sdepot delivery technology (Nano Precision Medical; Emeryville, Calif.)is contemplated. The technology utilizes a titania nanotube membranethat produces zero-order release rates of macromolecules, such asprotein and peptide therapeutics. The biocompatible membrane is housedin a small, subcutaneous implant that provides long-term (e.g., up toone year), constant-rate delivery of therapeutic macromolecules. Thetechnology is currently being evaluated, e.g., for the delivery of GLP-1agonists for the treatment of Type II diabetes.

The pharmaceutical compositions can be in the form of a sterileinjectable aqueous or oleaginous suspension. This suspension can beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents mentioned herein. The sterileinjectable preparation can also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Acceptable diluents,solvents and dispersion media that can be employed include water,Ringer's solution, isotonic sodium chloride solution, Cremophor EL™(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS), ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol), and suitable mixtures thereof. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil can be employed including synthetic mono- ordiglycerides. Moreover, fatty acids such as oleic acid find use in thepreparation of injectables. Prolonged absorption of particularinjectable formulations can be achieved by including an agent thatdelays absorption (e.g., aluminum monostearate or gelatin).

The pharmaceutical compositions containing the active ingredient can bein a form suitable for oral use, for example, as tablets, capsules,troches, lozenges, aqueous or oily suspensions, dispersible powders orgranules, emulsions, hard or soft capsules, or syrups, solutions,microbeads or elixirs. Pharmaceutical compositions intended for oral usecan be prepared according to any method known to the art for themanufacture of pharmaceutical compositions, and such compositions cancontain one or more agents such as, for example, sweetening agents,flavoring agents, coloring agents and preserving agents in order toprovide pharmaceutically elegant and palatable preparations. Tablets,capsules and the like contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients which are suitable forthe manufacture of tablets. These excipients can be, for example,diluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc.

The tablets, capsules and the like suitable for oral administration canbe uncoated or coated by known techniques to delay disintegration andabsorption in the gastrointestinal tract and thereby provide a sustainedaction. For example, a time-delay material such as glyceryl monostearateor glyceryl distearate can be employed. They can also be coated bytechniques known in the art to form osmotic therapeutic tablets forcontrolled release. Additional agents include biodegradable orbiocompatible particles or a polymeric substance such as polyesters,polyamine acids, hydrogel, polyvinyl pyrrolidone, polyanhydrides,polyglycolic acid, ethylene-vinylacetate, methylcellulose,carboxymethylcellulose, protamine sulfate, or lactide/glycolidecopolymers, polylactide/glycolide copolymers, or ethylenevinylacetatecopolymers in order to control delivery of an administered composition.For example, the oral agent can be entrapped in microcapsules preparedby coacervation techniques or by interfacial polymerization, by the useof hydroxymethylcellulose or gelatin-microcapsules or poly(methylmethacrolate) microcapsules, respectively, or in a colloid drugdelivery system. Colloidal dispersion systems include macromoleculecomplexes, nano-capsules, microspheres, microbeads, and lipid-basedsystems, including oil-in-water emulsions, micelles, mixed micelles, andliposomes. Methods of preparing liposomes are described in, for example,U.S. Pat. Nos. 4,235,871, 4,501,728, and 4,837,028. Methods for thepreparation of the above-mentioned formulations will be apparent tothose skilled in the art.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate, kaolin ormicrocrystalline cellulose, or as soft gelatin capsules wherein theactive ingredient is mixed with water or an oil medium, for examplepeanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture thereof. Such excipients can besuspending agents, for example sodium carboxymethylcellulose,methylcellulose, hydroxy-propylmethylcellulose, sodium alginate,polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing orwetting agents, for example a naturally-occurring phosphatide (e.g.,lecithin), or condensation products of an alkylene oxide with fattyacids (e.g., polyoxy-ethylene stearate), or condensation products ofethylene oxide with long chain aliphatic alcohols (e.g., forheptadecaethyleneoxycetanol), or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol (e.g.,polyoxyethylene sorbitol monooleate), or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides (e.g., polyethylene sorbitan monooleate). The aqueoussuspensions can also contain one or more preservatives.

Oily suspensions can be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents can be added to provide a palatable oralpreparation.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified herein.

The pharmaceutical compositions of the present disclosure can also be inthe form of oil-in-water emulsions. The oily phase can be a vegetableoil, for example olive oil or arachis oil, or a mineral oil, forexample, liquid paraffin, or mixtures of these. Suitable emulsifyingagents can be naturally-occurring gums, for example, gum acacia or gumtragacanth; naturally-occurring phosphatides, for example, soy bean,lecithin, and esters or partial esters derived from fatty acids; hexitolanhydrides, for example, sorbitan monooleate; and condensation productsof partial esters with ethylene oxide, for example, polyoxyethylenesorbitan monooleate.

Formulations can also include carriers to protect the compositionagainst rapid degradation or elimination from the body, such as acontrolled release formulation, including implants, liposomes,hydrogels, prodrugs and microencapsulated delivery systems. For example,a time delay material such as glyceryl monostearate or glyceryl stearatealone, or in combination with a wax, can be employed.

The present disclosure contemplates the administration of thepolypeptides in the form of suppositories for rectal administration ofthe drug. The suppositories can be prepared by mixing the drug with asuitable non-irritating excipient which is solid at ordinarytemperatures but liquid at the rectal temperature and will thereforemelt in the rectum to release the drug. Such materials include, but arenot limited to, cocoa butter and polyethylene glycols.

The polypeptides contemplated by the present disclosure can be in theform of any other suitable pharmaceutical composition (e.g., sprays fornasal or inhalation use) currently known or developed in the future.

The concentration of a polypeptide or fragment thereof in a formulationcan vary widely (e.g., from less than about 0.1%, usually at or at leastabout 2% to as much as 20% to 50% or more by weight) and will usually beselected primarily based on fluid volumes, viscosities, andsubject-based factors in accordance with, for example, the particularmode of administration selected.

Routes of Administration

The present disclosure contemplates the administration of the disclosedpolypeptides, and compositions thereof, in any appropriate manner.Suitable routes of administration include parenteral (e.g.,intramuscular, intravenous, subcutaneous (e.g., injection or implant),intraperitoneal, intracisternal, intraarticular, intraperitoneal,intracerebral (intraparenchymal) and intracerebroventricular), oral,nasal, vaginal, sublingual, intraocular, rectal, topical (e.g.,transdermal), sublingual and inhalation.

Depot injections, which are generally administered subcutaneously orintramuscularly, can also be utilized to release the polypeptidesdisclosed herein over a defined period of time. Depot injections areusually either solid- or oil-based and generally comprise at least oneof the formulation components set forth herein. One of ordinary skill inthe art is familiar with possible formulations and uses of depotinjections.

Regarding antibodies, in an exemplary embodiment an antibody or antibodyfragment of the present disclosure is stored at 10 mg/ml in sterileisotonic aqueous saline solution for injection at 4° C. and is dilutedin either 100 ml or 200 ml 0.9% sodium chloride for injection prior toadministration to the subject. The antibody is administered byintravenous infusion over the course of 1 hour at a dose of between 0.2and 10 mg/kg. In other embodiments, the antibody is administered byintravenous infusion over a period of between 15 minutes and 2 hours. Instill other embodiments, the administration procedure is viasubcutaneous bolus injection.

Combination Therapy

The present disclosure contemplates the use of the FGF19 variantpolypeptides identified herein in combination with one or more activetherapeutic agents or other prophylactic or therapeutic modalities. Insuch combination therapy, the various active agents frequently havedifferent mechanisms of action. Such combination therapy can beespecially advantageous by allowing a dose reduction of one or more ofthe agents, thereby reducing or eliminating the adverse effectsassociated with one or more of the agents; furthermore, such combinationtherapy can have a synergistic therapeutic or prophylactic effect on theunderlying disease, disorder, or condition.

As used herein, “combination” is meant to include therapies that can beadministered separately, for example, formulated separately for separateadministration (e.g., as can be provided in a kit), and therapies thatcan be administered together in a single formulation (i.e., a“co-formulation”). Combinations of the polypeptides identified using themethods and models described herein with one or more active therapeuticagents or other prophylactic or therapeutic modalities can beadministered or applied sequentially (e.g., where one agent isadministered prior to one or more other agents) or simultaneously (e.g.,where two or more agents are administered at or about the same time).Regardless of whether the two or more agents are administeredsequentially or simultaneously, they are considered to be administeredin combination for purposes of the present disclosure.

Accordingly, methods and uses of the polypeptides identified through useof the methods and models described herein can be practiced prior to,substantially contemporaneously with or following another treatment, andcan be supplemented with other forms of therapy. Supplementary therapiesinclude other glucose lowering and/or weigh loss treatments, such asinsulin, an insulin sensitivity enhancer and other drug treatments, achange in diet (low sugar, fats, etc.), weight loss surgery- (reducingstomach volume by gastric bypass, gastrectomy), gastric banding, gastricballoon, gastric sleeve, etc.

The present disclosure contemplates combination therapy with numerousagents (and classes thereof), including 1) insulin, insulin mimetics andagents that entail stimulation of insulin secretion, includingsulfonylureas (e.g., chlorpropamide, tolazamide, acetohexamide,tolbutamide, glyburide, glimepiride, glipizide) and meglitinides (e.g.,repaglinide (PRANDIN) and nateglinide (STARLIX)); 2) biguanides (e.g.,metformin (GLUCOPHAGE)) and other agents that act by promoting glucoseutilization, reducing hepatic glucose production and/or diminishingintestinal glucose output; 3) alpha-glucosidase inhibitors (e.g.,acarbose and miglitol) and other agents that slow down carbohydratedigestion and consequently absorption from the gut and reducepostprandial hyperglycemia; 4) thiazolidinediones (e.g., rosiglitazone(AVANDIA), troglitazone (REZULIN), pioglitazone (ACTOS), glipizide,balaglitazone, rivoglitazone, netoglitazone, troglitazone, englitazone,ciglitazone, adaglitazone, darglitazone that enhance insulin action(e.g., by insulin sensitization), thus promoting glucose utilization inperipheral tissues; 5) glucagon-like-peptides including DPP-IVinhibitors (e.g., vildagliptin (GALVUS) and sitagliptin (JANUVIA)) andGlucagon-Like Peptide-1 (GLP-1) and GLP-1 agonists and analogs (e.g.,exenatide (BYETTA and ITCA 650 (an osmotic pump inserted subcutaneouslythat delivers an exenatide analog over a 12-month period; Intarcia,Boston, Mass.)); 6) and DPP-IV-resistant analogues (incretin mimetics),PPAR gamma agonists, dual-acting PPAR agonists, pan-acting PPARagonists, PTP1B inhibitors, SGLT inhibitors, insulin secretagogues, RXRagonists, glycogen synthase kinase-3 inhibitors, immune modulators,beta-3 adrenergic receptor agonists, 11beta-HSD1 inhibitors, and amylinanalogues.

Furthermore, the present disclosure contemplates combination therapywith agents and methods for promoting weight loss, such as agents thatstimulate metabolism or decrease appetite, and modified diets and/orexercise regimens to promote weight loss. Appetite suppression drugs arewell known and can be used in combination with the methods providedherein.

The FGF19 variant polypeptides of the present disclosure can be used incombination with one or more other agent in any manner appropriate underthe circumstances. In one embodiment, treatment with the at least oneactive agent and at least one polypeptide of the present disclosure ismaintained over a period of time. In another embodiment, treatment withthe at least one active agent is reduced or discontinued (e.g., when thesubject is stable), while treatment with the polypeptide of the presentdisclosure is maintained at a constant dosing regimen. In a furtherembodiment, treatment with the at least one active agent is reduced ordiscontinued (e.g., when the subject is stable), while treatment withthe polypeptide of the present disclosure is reduced (e.g., lower dose,less frequent dosing or shorter treatment regimen). In yet anotherembodiment, treatment with the at least one active agent is reduced ordiscontinued (e.g., when the subject is stable), and treatment with thepolypeptide of the present disclosure is increased (e.g., higher dose,more frequent dosing or longer treatment regimen). In yet anotherembodiment, treatment with the at least one active agent is maintainedand treatment with the polypeptide of the present disclosure is reducedor discontinued (e.g., lower dose, less frequent dosing or shortertreatment regimen). In yet another embodiment, treatment with the atleast one active agent and treatment with the polypeptide of the presentdisclosure are reduced or discontinued (e.g., lower dose, less frequentdosing or shorter treatment regimen).

Dosing

The polypeptides of the present disclosure can be administered to asubject in an amount that is dependent upon, for example, the goal ofthe administration (e.g., the degree of resolution desired); the age,weight, sex, and health and physical condition of the subject to betreated; the nature of the polypeptide, and/or formulation beingadministered; the route of administration; and the nature of thedisease, disorder, condition or symptom thereof (e.g., the severity ofthe dysregulation of glucose/insulin and the stage of the disorder). Thedosing regimen can also take into consideration the existence, nature,and extent of any adverse effects associated with the agent(s) beingadministered. Effective dosage amounts and dosage regimens can readilybe determined from, for example, safety and dose-escalation trials, invivo studies (e.g., animal models), and other methods known to theskilled artisan.

In general, dosing parameters dictate that the dosage amount be lessthan an amount that could be irreversibly toxic to the subject (i.e.,the maximum tolerated dose, “MTD”) and not less than an amount requiredto produce a measurable effect on the subject. Such amounts aredetermined by, for example, the pharmacokinetic and pharmacodynamicparameters associated with absorption, distribution, metabolism, andexcretion (“ADME”), taking into consideration the route ofadministration and other factors.

An effective dose (ED) is the dose or amount of an agent that produces atherapeutic response or desired effect in some fraction of the subjectstaking it. The “median effective dose” or ED50 of an agent is the doseor amount of an agent that produces a therapeutic response or desiredeffect in 50% of the population to which it is administered. Althoughthe ED50 is commonly used as a measure of reasonable expectance of anagent's effect, it is not necessarily the dose that a clinician mightdeem appropriate taking into consideration all relevant factors. Thus,in some situations the effective amount is more than the calculatedED50, in other situations the effective amount is less than thecalculated ED50, and in still other situations the effective amount isthe same as the calculated ED50.

In addition, an effective dose of the polypeptides of the presentdisclosure can be an amount that, when administered in one or more dosesto a subject, produces a desired result relative to a healthy subject.For example, an effective dose can be one that, when administered to asubject having elevated plasma glucose and/or plasma insulin, achieves adesired reduction relative to that of a healthy subject by at leastabout 10%, at least about 20%, at least about 25%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, or more than 80%.

An appropriate dosage level will generally be about 0.001 to 100 mg/kgof patient body weight per day, which can be administered in single ormultiple doses. In some embodiments, the dosage level will be about 0.01to about 25 mg/kg per day, and in other embodiments about 0.05 to about10 mg/kg per day. A suitable dosage level can be about 0.01 to 25 mg/kgper day, about 0.05 to 10 mg/kg per day, or about 0.1 to 5 mg/kg perday. Within this range, the dosage can be 0.005 to 0.05, 0.05 to 0.5 or0.5 to 5.0 mg/kg per day.

For administration of an oral agent, the compositions can be provided inthe form of tablets, capsules and the like containing from 1.0 to 1000milligrams of the active ingredient, particularly 1.0, 3.0, 5.0, 10.0,15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0,500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the activeingredient. The polypeptides can be administered on a regimen of, forexample, 1 to 4 times per day, and often once or twice per day.

The dosage of the polypeptides of the present disclosure can be repeatedat an appropriate frequency, which can be in the range of once per dayto once every three months, depending on the pharmacokinetics of thepolypeptides (e.g. half-life) and the pharmacodynamic response (e.g. theduration of the therapeutic effect of the polypeptide). In someembodiments where the polypeptide is an antibody or a fragment thereof,or a polypeptide or variants thereof, dosing is frequently repeatedbetween once per week and once every 3 months. In other embodiments,such polypeptides are administered approximately once per month.

In certain embodiments, the dosage of the disclosed polypeptides iscontained in a “unit dosage form.” The phrase “unit dosage form” refersto physically discrete units, each unit containing a predeterminedamount of a polypeptide of the present disclosure, either alone or incombination with one or more additional agents, sufficient to producethe desired effect. It will be appreciated that the parameters of a unitdosage form will depend on the particular agent and the effect to beachieved. Exemplary unit doses can range from about 25-250; 250-500;500-1,000; 1,000-2,500; 2,500-5,000; 5,000-25,000; or 25,000-50,000 ng;or from about 25-250; 250-500; 500-1,000; 1,000-2,500; 2,500-5,000;5,000-25,000; 25,000-50,000 mg; or from about 25-250; 250-500;500-1,000; 1000-2,500; 2,500-5,000; 5,000-25,000; or 25,000-50,000 mg.

Single or multiple doses can be administered, for example, multipletimes per day, on consecutive days, alternating days, weekly orintermittently (e.g., twice per week, once every 1, 2, 3, 4, 5, 6, 7 or8 weeks, or once every 2, 3, 4, 5 or 6 months).

Kits

The present disclosure also contemplates kits comprising the disclosedpolypeptides, and pharmaceutical compositions thereof. The kits aregenerally in the form of a physical structure housing variouscomponents, as described below, and can be utilized, for example, inpracticing the methods described above (e.g., administration of apolypeptide to a subject in need of restoring glucose homeostasis).

A kit can include one or more of the polypeptides disclosed herein(provided in, e.g., a sterile container), which can be in the form of apharmaceutical composition suitable for administration to a subject. Thepolypeptides can be provided in a form that is ready for use or in aform requiring, for example, reconstitution or dilution prior toadministration. When the polypeptides are in a form that needs to bereconstituted by a user, the kit can also include buffers,pharmaceutically acceptable excipients, and the like, packaged with orseparately from the polypeptides. When combination therapy iscontemplated, the kit can contain the several agents separately or theycan already be combined in the kit. Each component of the kit can beenclosed within an individual container and all of the variouscontainers can be within a single package. A kit of the presentdisclosure can be designed for conditions necessary to properly maintainthe components housed therein (e.g., refrigeration or freezing).

A kit can contain a label or packaging insert including identifyinginformation for the components therein and instructions for their use(e.g., dosing parameters, clinical pharmacology of the activeingredient(s), including mechanism of action, pharmacokinetics andpharmacodynamics, adverse effects, contraindications, etc.). Labels orinserts can include manufacturer information such as lot numbers andexpiration dates. The label or packaging insert can be, e.g., integratedinto the physical structure housing the components, contained separatelywithin the physical structure, or affixed to a component of the kit(e.g., an ampoule, tube or vial). Exemplary instructions include thosefor reducing or lowering blood glucose, treatment of hyperglycemia,treatment of diabetes, etc. with the disclosed polypeptides, andpharmaceutical compositions thereof.

Labels or inserts can additionally include, or be incorporated into, acomputer readable medium, such as a disk (e.g., hard disk, card, memorydisk), optical disk such as CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape,or an electrical storage media such as RAM and ROM or hybrids of thesesuch as magnetic/optical storage media, FLASH media or memory-typecards. In some embodiments, the actual instructions are not present inthe kit, but means for obtaining the instructions from a remote source,e.g., via the internet, are provided.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed herein.

In case of conflict, the specification, including definitions, willcontrol. As used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a peptide sequence”or a “treatment,” includes a plurality of such sequences, treatments,and so forth. It is further noted that the claims can be drafted toexclude any optional element. As such, this statement is intended toserve as antecedent basis for use of such exclusive terminology such as“solely,” “only” and the like in connection with the recitation of claimelements, or use of a “negative” limitation.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges can independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

As used herein, numerical values are often presented in a range formatthroughout this document. The use of a range format is merely forconvenience and brevity and should not be construed as an inflexiblelimitation on the scope of the invention unless the context clearlyindicates otherwise. Accordingly, the use of a range expressly includesall possible subranges, all individual numerical values within thatrange, and all numerical values or numerical ranges including integerswithin such ranges and fractions of the values or the integers withinranges, unless the context clearly indicates otherwise. Thisconstruction applies regardless of the breadth of the range and in allcontexts throughout this patent document. Thus, for example, referenceto a range of 90-100% includes 91-99%, 92-98%, 93-95%, 91-98%, 91-97%,91-96%, 91-95%, 91-94%, 91-93%, and so forth. Reference to a range of90-100% also includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, etc., as wellas 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%,92.5%, etc., and so forth. In addition, reference to a range of 1-3,3-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90,90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170,170-180, 180-190, 190-200, 200-225, 225-250 includes 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. In a furtherexample, reference to a range of 25-250, 250-500, 500-1000, 1000-2500,2500-5000, 5000-25,000, or 5000-50,000 includes any numerical value orrange within or encompassing such values, e.g., 25, 26, 27, 28, 29 . . .250, 251, 252, 253, 254 . . . 500, 501, 502, 503, 504 . . . , etc. Theuse of a series of ranges includes combinations of the upper and lowerranges to provide another range. This construction applies regardless ofthe breadth of the range and in all contexts throughout this patentdocument. Thus, for example, reference to a series of ranges such as5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, 100-150, includesranges such as 5-20, 5-30, 5-40, 5-50, 5-75, 5-100, 5-150, and 10-30,10-40, 10-50, 10-75, 10-100, 10-150, and 20-40, 20-50, 20-75, 20-100,20-150, and so forth.

For the sake of conciseness, certain abbreviations are used herein. Oneexample is the single letter abbreviation to represent amino acidresidues. The amino acids and their corresponding three letter andsingle letter abbreviations are as follows:

alanine Ala (A) arginine Arg (R) asparagine Asn (N) aspartic acid Asp(D) cysteine Cys (C) glutamic acid Glu (E) glutamine Gln (Q) glycine Gly(G) histidine His (H) isoleucine Ile (I) leucine Leu (L) lysine Lys (K)methionine Met (M) phenylalanine Phe (F) proline Pro (P) serine Ser (S)threonine Thr (T) tryptophan Trp (W) tyrosine Tyr (Y) valine Val (V)

The invention is generally disclosed herein using affirmative languageto describe the numerous embodiments. The invention also specificallyincludes embodiments in which particular subject matter is excluded, infull or in part, such as substances or materials, method steps andconditions, protocols, procedures, assays or analysis. Thus, even thoughthe invention is generally not expressed herein in terms of what theinvention does not include, aspects that are not expressly included inthe invention are nevertheless disclosed herein.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, the descriptions in the Experimental section are intendedto illustrate but not limit the scope of invention described in theclaims.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature, etc.), but someexperimental errors and deviations should be accounted for.

Unless indicated otherwise, parts are parts by weight, molecular weightis weight average molecular weight, temperature is in degrees Celsius (°C.), and pressure is at or near atmospheric. Standard abbreviations areused, including the following: bp=base pair(s); kb=kilobase(s);pl=picoliter(s); s or sec=second(s); min=minute(s); h or hr=hour(s);aa=amino acid(s); kb=kilobase(s); nt=nucleotide(s); ng=nanogram;μg=microgram; mg=milligram; g=gram; kg=kilogram; dl or dL=deciliter; μlor μL=microliter; ml or mL=milliliter; 1 or L=liter; μM=micromolar;mM=millimolar; M=molar; kDa=kilodalton; i.m.=intramuscular(ly);i.p.=intraperitoneal(ly); s.c.=subcutaneous(ly); bid=twice daily;HPLC=high performance liquid chromatography; BW=body weight; U=unit;ns=not statistically significant; PG=fasting plasma glucose; FPI=fastingplasma insulin; ITT=insulin tolerance test; PTT=pyruvate tolerance test;oGTT=oral glucose tolerance test; GSIS=glucose-stimulated insulinsecretion; AAV=adneno-associated virus; PBS=phosphate-buffered saline;PCR=polymerase chain reaction; NHS=N-Hydroxysuccinimide; DMEM=Dulbeco'sModification of Eagle's Medium; GC=genome copy;EDTA=ethylenediaminetetraacetic acid; FGF19CF=FGF19 with FLAG-tag at theC-terminus; GFP=green fluorescent protein; ELISA=enzyme-linkedimmunosorbance assay; ANOVA=analysis of variance; SEM=standard error ofthe mean.

Example 1 Materials and Methods for Examples 2-5

The following methods and materials were used in Examples 2-5 below.

Animals.

db/db mice (The Jackson Laboratory; Bar Harbor, Me.), approximately 15weeks old mice and weighing approximately 36-48 g at initiation oftreatment, were kept in accordance with welfare guidelines undercontrolled light (12-hour light and 12-hour dark cycle, dark 6:30p.m.-6:30 a.m.), temperature (22±4° C.) and humidity (50%±20%)conditions. Mice had free access to autoclaved distilled water and werefed ad libitum a commercial diet (Harlan Laboratories, Indianapolis,Ind., Irradiated 2018 Teklad Global 18% Protein Rodent Diet) containing18 kcal % fat, 24 kcal % protein and 58 kcal % carbohydrate. All animalstudies were approved by the NGM Institutional Animal Care and UseCommittee.

Nucleic Acid and Amino Acid Sequences.

FGF19 ORF (cDNA of ORF encoding hFGF19 (GenBank Accession No. NM005117.2) and protein sequence encoded thereby (GenBank Accession No.NP_005108.1)) was amplified via PCR using recombinant DNA (cDNA)prepared from human small intestinal tissue. PCR reagent kits withPhusion® high-fidelity DNA polymerase ((F-530L; New England BioLabs;Ipswich, Mass.) were used with the following primers: forward PCRprimer: 5′ CCGACTAGTCACCatgcggagcgggtgtgtgg (SEQ ID NO:40), and reversePCR primer: 5′ ATAAGAATGCGGCCGCTTACTTCTCAAAGCTGGGACTCCTC (SEQ ID NO:41).

Amplified DNA fragment was digested with Spe I and Not I (therestriction sites were included in the 5′ or 3′ PCR primers,respectively) and was then ligated with AAV transgene vectors that hadbeen digested with the same restriction enzymes. The vector used forexpression contained a selectable marker and an expression cassettecomposed of a strong eukaryotic promoter 5′ of a site for insertion ofthe cloned coding sequence, followed by a 3′ untranslated region andbovine growth hormone polyadenylation tail. The expression construct wasalso flanked by internal terminal repeats at the 5′ and 3′ ends.

Production and Purification of AAV Encoding FGF19 and FGF19 Variants.

AAV293 cells (Agilent Technologies, Santa Clara, Calif.) were culturedin Dulbeco's Modification of Eagle's Medium (DMEM, Mediatech, Herndon,Va.) supplemented with 10% fetal bovine serum and 1×antibiotic-antimycotic solution (Mediatech). The cells were plated at50% density on Day 1 in 150-mm cell culture plates and transfected onDay 2, using calcium phosphate precipitation method, with the followingthree plasmids (20 μg/plate of each): i) AAV transgene plasmid, ii)pHelper plasmids (Agilent Technologies), and iii) AAV2/9 plasmid(Rabinowitz et al. 2002). Forty-eight hours after transfection, thecells were scraped off the plates, pelleted by centrifugation at 3000×gand re-suspended in buffer containing 20 mM Tris pH 8.5, 100 mM NaCl and1 mM MgCl₂. The suspension was frozen in an alcohol dry ice bath and wasthen thawed in 37° C. water bath; the freeze-thaw cycle was repeatedthree times. Benzonase® (Sigma-Aldrich; St. Louis, Mo.) was added to 50units/mL and deoxycholate was added to a final concentration of 0.25%.After incubation at 37° C. for 30 minutes, cell debris was pelleted bycentrifugation at 5000×g for 20 minutes. Viral particles in thesupernatant were purified using a discontinued iodixanal (Sigma-Aldrich)gradient as previously described (Zolotukhin et al., (2002)Endocrinology 143(5):1741-47. The viral stock was concentrated usingVivaspin® 20 (molecular weight (MW) cutoff 100,000 Da, Sartorius StedimBiotech; Aubagne, France) and re-suspended in phosphate-buffered saline(PBS) with 10% glycerol and stored at −80° C.

To determine the viral genome copy (GC) number, 2 μL of viral stock wasincubated in 6 μL of solution containing 50 units/mL Benzonase, 50 mMTris-HCl pH 7.5, 10 mM MgCl₂, and 10 mM CaCl₂ at 37° C. for 30 minutes.Afterwards, 15 μL of the solution containing 2 mg/mL of Proteinase K,0.5% SDS, and 25 mM EDTA were added and the mixture was incubated for anadditional 20 minutes at 55° C. to release viral DNA. Viral DNA wascleaned with mini DNeasy® Kit (Qiagen; Valencia, Calif.) and eluted with40 μL of water. Viral GC was determined using quantitative PCR. Viralstock was diluted with saline to the desirable GC/mL and the workingsolution (200 μL) was injected into mice via a tail vein.

Blood Glucose Assay.

Blood samples were collected from individual non-fasted animals by tailsnip, and plasma glucose levels were measured using a glucometer(Accu-Chek® instruments; Roche Diagnostics, Indianapolis, Ind.)following manufacturer's instruction.

Serum FGF19 and FGF19 Variant Exposure Level Assay.

Whole blood (˜50 μl/mouse) from mouse tail snips was collected intoplain capillary tubes (BD Clay Adams SurePrep™, Becton Dickenson;Sparks, Md.). Serum and blood cells were separated by centrifugation for10 mins at 10,000 rpm, 4° C. in an Autocrit™ Ultra 3 centrifuge (BectonDickinson) and immediately frozen at −80° C. Levels of FGF19 and FGF19variants were measured in serum using a commercially available ELISA(Biovendor; Asheville, N.C.) following the manufacturer's instructions.Human FGF19 was used as the standard and relative concentrations of M70were determined. Relative concentrations of other FGF19 variants can bedetermined accordingly.

Fat Mass and Lean Mass Measurements.

Un-anesthetized animals were placed individually in a plastic holder andbody composition determined using NMR-MRI (whole body compositionanalyzer, EchoMRI™, Houston, Tex.). Fat mass, lean mass, and watercontent (data not presented) were recorded. The entire procedure did notexceed 2 minutes for each animal.

Gross Liver Nodule Assessment.

Twenty-four weeks after AAV injection, animals were euthanized andindividual livers were examined for gross nodule formation. The numberof visible liver nodules (>2 mm in diameter) were counted and recorded.

Statistical Analysis.

All results were expressed as the mean±standard error of the mean (SEM).One-way ANOVA followed by Dunnett's post-test was used to compare datafrom multiple groups (GraphPad Prism®; San Diego, Calif.). Whenindicated, unpaired Student's t-test was used to compare two treatments.Two-way ANOVA followed by Bonferroni's post-test was use to comparemultiple groups for time-course studies. A p-value of 0.05 or smallerwas considered statistically significant.

Example 2 Plasma FGF19 Levels in db/db Mice Following Gene Delivery

A 24-week study was conducted in order to evaluate whether the FGF19variant M70 was able to block FGF19-induced tumorigenicity in db/dbmice. As an alternative to conventional methods of delivery, AAV wasused in this example (and examples 2-4 that follow) as the vehicle todeliver and express exogenous genes of interest in mice and enablecontinuous, persistent and systemic exposure to proteins encoded bythose transgenes.

Prior to gene delivery, mice were sorted into six groups (5 malemice/group) as set forth in Table 1, and blood glucose and body weightmeasurements were recorded for each mouse.

TABLE 1 Dose Level Volume Group AAV Construct (AAV) (mL/mouse)Descriptor 1 Saline 0 0.2 Control 2 GFP 3e11 0.2 AAV-Control 3FGF19-flag 3e9 0.2 FGF19 Low Dose 4 FGF19-flag 3e10 0.2 FGF19 High Dose5 M70 3e11 0.2 (total) M70/FGF19 FGF19-flag 3e9 Low Dose 6 M70 3e11 0.2(total) M70/FGF19 FGF19-flag 3e10 High Dose

At week 0, mice were injected either with 0.2 mL saline or 0.2 mL of oneof the AAV constructs from Groups 2-6. At weeks 3 and 5, blood glucoseand body weight measurements were again recorded for each mouse inGroups 1-6.

Five weeks after gene delivery, FGF19 concentrations were measured insera isolated from mice injected either with saline (Group 1) or AAVconstructs (Groups 2-6). Since the ELISA used to measure drugconcentrations was unable to accurately distinguish between FGF19 andM70, plasma levels determined for Groups 5 and 6 represent the totalplasma concentrations of both proteins.

The results are set forth in FIG. 2. FGF19 levels detected in micereceiving low (3e9; Group 3) and high (3e10; Group 4) doses ofrecombinant FGF19-flag virus were proportional to AAV dose (1.4±0.5ng/mL and 93.6±12.6 ng/mL, respectively). In mice injected with both theFGF19-flag and M70 transgenes, the M70 virus (3e11) was present ateither 100- or 10-fold excess compared with the FGF19-flag constructalone. As a result of co-injecting the two transgenes, high serum levelsof FGF19 were detected at both low and high doses of FGF19-flag(734.0±61.1 ng/mL (Group 5) and 453.4±169.4 ng/mL (Group 6),respectively), representing contributions from the expression of bothM70 and FGF19-flag. In contrast, FGF19 was undetectable in samplesisolated from db/db mice injected with either saline or AAV-GFP.

At week 23, blood glucose and body weight measurements were againrecorded for each mouse. Twenty-four weeks after gene delivery, allanimals were euthanized and subjected to necropsy.

Example 3 FGF19-Mediated Formation of Gross Hepatic Nodules in db/dbMice in the Absence and Presence of FGF19 Variant M70

Using the euthanized animals from Example 2, livers from individual micewere examined, and the numbers of visible liver nodules were determined.The results are set forth in FIG. 3. References to Group numbers referto Table 1.

As depicted in FIG. 3, ectopic expression of FGF19-flag in the db/dbmouse model promoted the formation of multiple, large, raised nodulesprotruding from the liver surface at both low (3e9; Group 3)) and high(3e10; Group 4) viral doses (2.4±1.4 lesions per liver and 7.8 lesionsper liver, respectively). By comparison, livers isolated from miceexpressing both FGF19-flag and M70 were completely free of hepaticnodules (Group 5 and Group 6). Results are expressed as the mean and SEMfor all animals within the same study group. It should be noted that thec-Flag component did not impact FGF19's tumorogenic effects, though itcan impact FGF19's antidiabetic effects.

Ectopic expression of FGF19-flag promotes the formation of hepaticnodules in db/db mice at serum concentrations as low as 1 ng/mL.However, FGF19-mediated tumorigenesis, as evidenced by the appearance ofhepatic lesions, is completely suppressed when the FGF19-flag and M70transgenes are co-expressed in this model. These data suggest that notonly does the engineered FGF19 variant M70 lack the tumorigenicpotential in mice associated with the wild-type protein, but that it caneffectively interfere with the proliferative effects of the wild-typeprotein.

Example 4 Effects of Transgene Expression on Body Weight and Compositionin db/db Mice

As alluded to in Example 2, 15-week-old male db/db mice (n=5) wereinjected with 0.2 mL saline or recombinant AAV transgenes as indicatedin Table 1. Body weights were measured for each mouse prior to injection(week −1) and 3-, 5- and 23-weeks post-injection. The results, set forthin FIG. 4, are expressed as the mean of individual measurements from allanimals and SEM.

Transgenic db/db mice co-expressing M70 and FGF19-flag (Groups 5 and 6)showed significant reductions in body weight as compared with animalsdosed with saline (Group 1). Less dramatic effects on body weight wereobserved in mice expressing the FGF19-flag transgene, although thereductions appeared to be dose-dependent and were significant at weeks 3and 5 in animals injected with the higher dose (Groups 3 and 4).

Note that mice in both control groups (dosed either with saline(Group 1) or AAV-GFP (Group 2)), tended to show significant loss of massby the end of the study, compared with their maximum body weights inweeks 3 and 5 following gene delivery. The body weight loss in theseanimals is commonly associated with the severe hyperglycemia observed indb/db mice and the progression of type 2 diabetes during the course ofthe 24-week study.

The changes in body weight observed in mice co-expressing the FGF19-flagand M70 transgenes were reflected in reduced liver weights compared withthose harvested from animals in the saline group (data not shown);notably, the reduced organ size was directly proportional to the lowerbody weight in these mice. In contrast, the relative liver weight wasincreased in mice expressing FGF19-flag, although these changes weresimilarly not significant when normalized to body weight (data notshown)

In addition, the effects of treatment on body composition weredetermined 23-weeks post-injection using NMR-MRI. Consistent with theobserved reductions in body weight, ectopic co-expression of the M70 andFGF19-flag transgenes resulted in the loss of both fat mass and leanmass in db/db mice compared with mice treated with saline (data notshown). Expression of FGF19-flag had little effect on body compositionin db/db mice receiving either the low or high dose of the transgene(data not shown).

Example 5 Effects of Transgene Expression on Non-Fasted Blood Glucose indb/db Mice

As alluded to in Example 2, 15-week-old male db/db mice (n=5) wereinjected with 0.2 mL saline or recombinant AAV transgenes as indicatedin Table 1. Blood glucose was measured for each mouse prior to injection(week −1) and 3-, 5- and 23-weeks post-injection. The results, set forthin FIG. 5, are expressed as the mean of individual measurements from allanimals and SEM.

Transgenic db/db mice co-expressing M70 and FGF19-flag (Groups 5 and 6)showed significant reductions in blood glucose concentrations ascompared with control animals (Groups 1 and 2). Glucose levels werereduced rapidly in mice co-expressing the FGF19-flag and M70 transgenes,reaching plateau levels by approximately 3 weeks after gene delivery(160 and 141 mg/dL at the high dose (Group 6) and low dose (Group 5) ofFGF19-flag transgene, respectively). The blood glucose levels in miceexpressing FGF19-flag (Groups 3 and 4) were significantly lower than incontrol groups, and maintained at initial baseline levels (approximately400-450 mg/dL) during the course of the 24-week study. As previouslyindicated, although the c-Flag component did not impact FGF19'stumorogenic effects, it can impact FGF19's antidiabetic effects. The lowsystemic levels of FGF19-flag detected in these mice appear to providesome protection against the deteriorating glycemia observed in micetreated with saline or AAV-GFP, but fail to lower glucose levels belowthe baseline values.

As would be expected, no glucose-lowering was observed during the courseof the study following injection with saline (Group 1) or a controlvirus, AAV-GFP (Group 2). Of note, the blood glucose concentrationsdetermined by glucometer in the control groups (˜600 mg/dL) representthe upper limit of detection by the instrument and may underrepresentthe actual glucose concentration in these samples.

Example 6 Materials and Methods for Examples 7-11

The following methods and materials were used in Examples 7-16 below.

DNA Constructs.

Human FGF19 (NM_005117), human FGFR4 (NM_022963), mouse FGFR4(NM_008011), human KLB (NM_175737) and mouse KLB (NM_031180) cDNAs werepurchased from Genecopoeia. Mutations were introduced in the FGF19constructs using the QuickChange™ Site-Directed Mutagenesis kit(Stratagene).

Production and Purification of AAV Encoding FGF19 and FGF19 Variants.

AAV293 cells (Agilent Technologies, Santa Clara, Calif.) were culturedin Dulbeco's Modification of Eagle's Medium (DMEM, Mediatech, Herndon,Va.) supplemented with 10% fetal bovine serum and 1×antibiotic-antimycotic solution (Mediatech). The cells were plated at50% density on Day 1 in 150-mm cell culture plates and transfected onDay 2, using calcium phosphate precipitation method, with the followingthree plasmids (20 μg/plate of each): i) AAV transgene plasmid, ii)pHelper plasmids (Agilent Technologies), and iii) AAV2/9 plasmid(Rabinowitz et al. 2002). Forty-eight hours after transfection, thecells were scraped off the plates, pelleted by centrifugation at 3000×gand re-suspended in buffer containing 20 mM Tris pH 8.5, 100 mM NaCl and1 mM MgCl₂. The suspension was frozen in an alcohol dry ice bath and wasthen thawed in 37° C. water bath; the freeze-thaw cycle was repeatedthree times. Benzonase® (Sigma-Aldrich; St. Louis, Mo.) was added to 50units/mL and deoxycholate was added to a final concentration of 0.25%.After incubation at 37° C. for 30 minutes, cell debris was pelleted bycentrifugation at 5000×g for 20 minutes. Viral particles in thesupernatant were purified using a discontinued iodixanal (Sigma-Aldrich)gradient as previously described (Zolotukhin et al., (2002)Endocrinology 143(5):1741-47. The viral stock was concentrated usingVivaspin® 20 (molecular weight (MW) cutoff 100,000 Da, Sartorius StedimBiotech; Aubagne, France) and re-suspended in phosphate-buffered saline(PBS) with 10% glycerol and stored at −80° C.

To determine the viral genome copy (GC) number, 2 μL of viral stock wasincubated in 6 μL of solution containing 50 units/mL Benzonase, 50 mMTris-HCl pH 7.5, 10 mM MgCl₂, and 10 mM CaCl₂ at 37° C. for 30 minutes.Afterwards, 15 μL of the solution containing 2 mg/mL of Proteinase K,0.5% SDS, and 25 mM EDTA were added and the mixture was incubated for anadditional 20 minutes at 55° C. to release viral DNA. Viral DNA wascleaned with mini DNeasy® Kit (Qiagen; Valencia, Calif.) and eluted with40 μL of water. Viral GC was determined using quantitative PCR. Viralstock was diluted with saline to the desirable GC/mL and the workingsolution (200 μL) was injected into mice via a tail vein.

Animal Experiments.

All animal studies were approved by the Institutional Animal Care andUse Committee at NGM. Mice were housed in a pathogen-free animalfacility at 22° C. under controlled 12 hour light/12 hour dark cycle.All mice were kept on standard chow diet (Harlan Laboratories, Teklad2918) and autoclaved water ad libitum. Male mice were used unlessotherwise specified. C57BL/6J, FVB/NJ, BDF, ob/ob, and db/db mice werepurchased from Jackson Laboratory. Heterozygous rasH2 transgenic micewere obtained from Taconic. On Day −7, cohorts of 10-12 week old ob/obor db/db mice, or 6-8 week old C57BL/6J, FVB/NJ, BDF, or rasH2 mice,were randomized into treatment groups based on body weight. All animalsreceived a single 200 μL intravenous injection of 3×10¹¹ genome copiesof AAV via tail vein on Day 1. Body weights were recorded and blood wascollected via tail snip for measurement of serum FGF19 levels. Animalswere euthanized and livers were collected 24 or 52 weeks after dosingwith AAV.

Gross, Histological, and Immunohistochemical Analysis.

To determine the onset of liver changes in mice injected with AAV-FGF19,gross and histological evaluations were performed at designatedintervals throughout the course of a year. Body weight, liver weight,and liver tumor nodule numbers were recorded upon necropsy. For tumorscore calculation for FGF19 variants, tumor score=number of tumornodules on the entire surface of the liver expressing variant/number oftumor nodules on the entire surface of the liver expressing wild typeFGF19. Therefore FGF19-expressing mice were given a tumor score with anarbitrary value of 1. Formalin-fixed paraffin-embedded tissue sectionswere stained with hematoxylin and eosin (H & E) for histologicalassessment of hepatocytic hyperplasia, hypertrophy, or neoplasia. Whenindicated, liver sections were treated for antigen retrieval usingcitrate buffer (Vector Laboratories) and then incubated with 10 μg/mLanti-PCNA (Dako), anti-Ki67 (Dako), anti-glutamine synthetase(Thermofisher), or anti-β-catenin antibodies (Cell Signaling).Biotinylated secondary antibody, ABC-HRP reagent, and DAB colorimetricperoxidase substrate (Vector Laboratories) were used for detection. ForLacZ staining, livers were embedded in OCT and sectioned on Cryostat.Tissue sections were fixed in PBS containing 4% paraformaldehyde and 2%glutaraldehyde for 10 minutes and incubated with 1 mg/mL X-gal (Promega)in 5 mM potassium ferrocyanide and 5 mM potassium ferricyanide at 37° C.for 2 hours.

Luciferase Assays.

Rat L6 myoblasts were obtained from American Type Culture Collection(ATCC) and cultured in Dulbecco's Modified Eagle Medium (DMEM)supplemented with 10% fetal bovine serum (FBS) at 37° C. under 5% CO2.Cells in 96-well plates were transiently transfected with expressionvectors encoding mouse KLB, mouse FGFR4, GAL4-Elk-1 transcriptionalactivator (pFA2-Elk1, Stratagene), firefly luciferase reporter drivenGAL4 binding sites (pFR-luc, Stratagene), and Renilla luciferase(pRL-SV40, Promega), using FuGENE® 6 transfection reagent (Roche AppliedScience). The day after transfection, the cells were stimulated for 6hours with ligands in serum free media containing 20 μg/mL heparin(Sigma). Cells were lysed with lysis buffer (Promega) and luciferaseactivity was determined using Dual-Glo® Luciferase Assay System(Promega) and EnSpire® Plate Reader (Perkin Elmer). Firefly luciferaseactivity was normalized to the co-expressed Renilla luciferase activityand shown as mean±SEM of three replicates.

Cyp7a1 Expression in Primary Hepatocytes.

Primary hepatocytes from mouse, rat or human livers (Life Technologies)were plated on collagen I-coated 96-well plates (Becton Dickinson) andincubated overnight in Wiliams' E media supplemented with 100 nMdexamethasone and 0.25 mg/mL matrigel. Cells were treated withrecombinant FGF19 or M70 proteins for 24 hours (mouse or rathepatocytes) or 6 hours (human hepatocytes). Cyp7a1 expression in celllysates was determined by qRT-PCR analysis using QuantiTect multiplexqRT-PCR master mix (Qiagen) and premade primers and probes (LifeTechnologies; mouse Cyp7a1: Mm00484150_m1; rat Cyp7a1: Rn00564065_m1;human Cyp7a1: Hs00167982_m1). Reactions were performed in triplicates onApplied Biosystems 7900HT Sequence Detection System. Relative mRNAlevels were calculated by the comparative threshold cycle method using18S RNA (mouse and rat) or actin (human) as the internal standard.

In Vivo Signaling Analysis.

db/db mice (9-11 week old) (Jackson Laboratories) were givenintraperitoneal (i.p.) injections (1 mg/kg) of FGF19 or M70 recombinantproteins. Livers were collected 15 minutes, 2 hours or 4 hours afterinjection and snap frozen in liquid nitrogen. Frozen liver samples werehomogenized in RIPA lysis buffer (50 mM Tris pH7.5, 150 mM NaCl, 1% NP40and 0.5% sodium deoxycholic acid, 1 mM dithiothereitol, 1 mM PMSF, 2 mMsodium fluoride, and 2 mM sodium orthovanadate) containing proteaseinhibitors (Roche) and phosphatase inhibitors (Sigma). Equal amounts ofprotein (15 μg), as determined by BCA assay (Thermo Fisher), wereseparated on 4-20% polyacrylamide gels (Bio-Rad) and transferred tonitrocellulose membranes (Bio-Rad). Membranes were blocked in 5% non-fatdry milk in PBS/0.05% Tween 20 and incubated with antibodies to pSTAT3(Cell Signaling), STAT3 (Cell Signaling), or antibody cocktail I (CellSignaling). Bound antibodies were detected with horse radish peroxidase(HRP)-conjugated secondary reagents and visualized using Odyssey®scanner (Li-Cor Biotechnology).

Xenograft Experiments.

6-8 week old athymic nu/nu female mice (Charles River Laboratories) wereinjected subcutaneously in the flanks with 5×10⁶ cells (200 μL/mouse).Mice bearing tumors of similar volumes (˜100 mm³) were randomized intogroups and treated via one-time tail vein injection of 3×10¹¹ AAV-M70 ora control virus (AAV-GFP). Tumors were measured with an electroniccaliper and average tumor volume was calculated using the formula:(W2×L)/2, where W and L are the smaller and large diameter,respectively.

Statistical Analysis.

All results are expressed as the mean±standard error of the mean (SEM).One-way ANOVA followed by Dunnett's post-test was used to compare datafrom multiple groups (GraphPad Prism®). When indicated, unpairedStudent's t-test was used to compare two treatment groups. Two-way ANOVAfollowed by Bonferroni's post-test was used to compare multiple groupsfor time-course studies. A p-value of 0.05 or smaller was consideredstatistically significant.

Example 7 An AAV-Mediated Transgene System for Evaluation ofHepatocellular Tumorigenesis In Vivo

AAV-mediated gene delivery provides a means to achieve continuoustransgene expression without inflammatory responses that are commonlyassociated with other viral vectors (Zaiss et al., 2002, J. Virol. 76,4580-4590). Sustained expression of up to 1 year has been observed withthe AAV gene delivery method when introduced into adult mice (Rivera etal., 1999, PNAS 96, 8657-8662). The first AAV vector was recentlyapproved as a treatment for a genetic disorder in human (Wirth et al.,2013, Gene 525, 162-169).

In previously reported FGF19 transgenic model, FGF19 was ectopicallyexpressed in the skeletal muscle, a non-physiological site of FGF19expression (Inagaki et al., 2005, Cell Metabol. 2, 217-225; Nicholes etal., 2002, Amer. J. Pathol. 160, 2295-2307). Under pathologicalconditions such as cirrhosis or cholestasis, FGF19 expression is inducedin the liver (Desnoyers et al., 2008, Oncogene 27, 85-97; Hasegawa Y,2013, Hepatol. 58, 802A; Schaap et al., 2009, Hepatol. 49, 1228-1235).As an alternative to conventional methods of generating transgenic mice,FGF19 was introduced via AAV in 6-12 week old mice (FIG. 6A). Theprimary tissue of transgene expression is liver using this approach,with only marginal expression in heart and muscle (data not shown).90-100% transduction of hepatocytes and long-term gene expressionwithout toxicity following a single administration of AAV were observedas previously reported (Zincarelli et al., 2008, Mol. Ther. 16,1073-1080)(data not shown).

Multiple mouse strains were evaluated for latency and robustness ofFGF19-mediated liver tumor formation (Table 2). A control AAV virus(AAV-GFP, green fluorescent protein) did not produce any liver tumors(Table 2).

As shown in Table 2, FGF19 Promotes Hepatocarcinogenesis in MultipleMouse Models. Various strains of mice (6-12 week old) were injected with3×10¹¹ genome copies of AAV vectors encoding FGF19 or a control gene(GFP, green fluorescent protein). Tumor incidence was determined at 24or 52 weeks after AAV administration. n.d., not determined.

TABLE 2 FGF19 Control Mouse 24 52 24 52 strain weeks weeks weeks weeksC57BL6/J 0/5 4/5 (80%) 0/5 0/5 BDF 0/5 5/5 (100%) 0/5 0/5 FVB/N 0/5 3/5(60%) 0/5 0/5 ob/ob 3/5 (60%) n.d. n.d. n.d. db/db 5/5 (100%) n.d. n.d.n.d.

In general, mice injected with AAV-GFP exhibited similar phenotype assaline-injected animals (data not shown). For simplicity, only resultsfrom AAV-GFP-injected animals were shown as controls in the followingstudies.

Interestingly, the tumor latency varied depending upon the mouse geneticbackground. Mutations in leptin receptor are frequently found incirrhotic livers and are linked to HCC in human (Ikeda et al., 2014,Gastroenterol., 146:222-232; Wang et al., 2010, World J. Gastroenterol.16, 5801-5809). db/db mice, which have a genetic defect in leptinreceptor (Tartaglia et al., 1995, Cell 83, 1263-1271), provide aclinically relevant genetic context for evaluating candidateHCC-promoting genes. Indeed, among several mouse strains tested, db/dbmice exhibited the shortest latency and high tumor penetrance, with theappearance of multiple, large, raised tumor nodules protruding from theliver surface 24 weeks following AAV-FGF19 delivery (FIG. 6B).

Serum FGF19 levels reached ˜1 ng/ml, 1 week after single tail veininjection of 3×10¹¹ genome copies of AAV-FGF19 in db/db mice (FIG. 6C).No FGF19 was detected in mice injected with control virus. The highcirculating levels of FGF19 persisted throughout the 24-week studyperiod (FIG. 6C). Visible tumor nodules on the entire surface of theliver were counted (FIG. 6D). The maximum diameter of the liver tumornodules was recorded (FIG. 6D). Occasionally a few liver tumor noduleswere observed in db/db mice injected with control virus or saline,probably reflecting increased background in tumorigenesis in thisgenetic model (FIG. 6D and data not shown). A tumor score system wasestablished based on the multiplicity of liver tumor nodules asdescribed in the materials and methods (Example 6) (FIG. 6D).

Microscopic examination of classified the AAV-FGF19-induced in situliver tumors as solid HCCs, which resembled those reported inFGF19-transgenic animals (FIG. 6E). Cellular proliferative status,examined by immunohistochemical staining for Ki-67 and PCNA, indicatedthat the tumors were highly proliferative. Similar to what was observedin FGF19-transgenic mice, liver tumors in AAV-FGF19 mice were glutaminesynthetase-positive, suggestive of a pericentral origin (Nicholes etal., 2002, Amer. J. Pathol. 160, 2295-2307) (FIG. 6E). Liver tumors fromAAV-FGF19 mice also showed increased nuclear staining for β-catenin(FIG. 6E). Thus, the AAV-mediated transgene expression provides a robustsystem to evaluate FGF19-induced hepatocarcinogenesis in vivo.

Example 8 M70 is an Engineered, Tumor-Free FGF19 Variant

FGF19 and FGF21 belong to the same FGF subfamily, sharing 34% amino acididentity. Interestingly, unlike FGF19, FGF21 does not induce liver tumorformation in our AAV-mediated transgene models (data not shown). Inorder to identify structural elements that are crucial fortumorigenicity induced by FGF19, a number of chimeric constructs betweenFGF19 and FGF21 were generated by systematically swapping predictedsecondary structural elements including α-strands and β-helices (Table3). Table 3 shows chimeric constructs with amino acid sequences derivedfrom FGF19 or FGF21. Liver tumor formation was assessed 24 weeks afterAAV-mediated transgene expression. Secondary structural components(β-sheets and loops between β-sheets) of FGF19 are replacedsystematically. Constructs were individually introduced into db/db miceby AAV to assess their tumorigenic potential after 24 weeks ofcontinuous exposure. The N-terminal 10-20 amino acids of FGF19 wereidentified as being critical for tumorigenicity (Table 3).

TABLE 3 Amino Amino Acids Acids from from Tumor Name FGF19 FGF21 ScoreControl 0.00 ± 0.00 FGF19 R23-K216 1.00 ± 0.18 FGF21 H28-S208 0.00 ±0.00 FGF19 “end swap” variants: N-ter R23-R43 H28-R44 0.00 ± 0.00 C-terP170-K216 R162-S208 2.70 + 0.50 FGF19 “loop swap” variants: Loop-1S50-L56 D51-T56 0.59 + 0.29 Loop-2 R63-G613 R63-G66 2.55 + 0.34 Loop-3A71-A76 A71-P76 1.58 + 0.51 Loop-4 A86-T89 K86-V89 1.69 + 0.27 Loop-5G94-S97 G94-T97 0.90 + 0.17 Loop-6 A105-G107 P105-G107 1.00 + 0.17Loop-7 L112-S116 S112-D129 0.67 + 0.18 Loop-8 R127-D129 L127-D129 0.06 +0.03 Loop-9 S136-H139 S136-H139 0.79 + 0.10 Loop-10 V143-L162 L143-K1491.32 + 0.22 Loop-11 R1574-H164 R158-G168 1.24 + 0.38 FGF19 “sheet swap”variants: Sheet-1 R43-T49 R44-T50 0.32 + 0.14 Sheet-2 S57-I62 E57-I620.35 + 0.10 Sheet-3 V67-A71 T67-A71 1.78 + 0.14 Sheet-4 L80-V85 L80-L853.99 + 0.63 Sheet-5 T89-K93 V89-L93 0.38 + 0.05 Sheet-6 V98-G104S98-R104 0.73 + 0.17 Sheet:7 K108-G111 A108-G111 1.91 + 0.66 Sheet-8F122-R127 F122-L127 0.94 + 0.21 Sheet-9 G130-S136 G130-S136 1.17 + 0.22Sheet-10 R140-P142 G140-P142 2.00 + 0.41 Sheet-11 F165-M168 R158-A1610.38 + 0.31

Subsequently, additional constructs were generated by only alteringamino acids within this region (Table 4). Table 4 shows the structureactivity relationship analysis of FGF19 variants in the N-terminalregion. Amino acid changes from wild type FGF19 are underlined. Livertumor formation was assessed 24 weeks after transgene expression.

TABLE 4 Tumor Name N-terminal Sequence Score ControlRPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSS 0.00 ± 0.00 FGF19 1.00 ± 0.18FGF19 N-terminal SAR variants: N1 R-----DAGPHVHYGWGDPIRLRHLYTSGPHGLSS1.25 ± 0.30 N2 R----------VHYGWGDPIRLRHLYTSGPHGLSS 0.00 ± 0.00 SSLR-----DSSPLVHYGWGDPIRLRHLYTSGPHGLSS 0.00 ± 0.00 SSHR-----DSSPHVHYGWGDPIRLRHLYTSGPHGLSS 2.39 ± 0.93 SGLR-----DSGPLVHYGWGDPIRLRHLYTSGPHGLSS 0.68 ± 0.14 ASLR-----DASPLVHYGWGDPIRLRHLYTSGPHGLSS 1.09 ± 0.18 EDLR-----DEDPLVHYGWGDPIRLRHLYTSGPHGLSS 1.18 ± 0.45 EGLR-----DEGPLVHYGWGDPIRLRHLYTSGPHGLSS 1.15 ± 0.14 EDHR-----DEDPHVHYGWGDPIRLRHLYTSGPHGLSS 1.00 ± 0.36 EGHR-----DEGPHVHYGWGDPIRLRHLYTSGPHGLSS 1.44 ± 0.05 QGHR-----DQGPHVHYGWGDPIRLRHLYTSGPHGLSS 1.01 ± 0.16 QGLR-----DQGPLVHYGWGDPIRLRHLYTSGPHGLSS 0.68 ± 0.12 QSHR-----DQSPHVHYGWGDPIRLRHLYTSGPHGLSS 1.42 ± 0.20 ESHR-----DESPHVHYGWGDPIRLRHLYTSGPHGLSS 1.22 ± 0.31 QSLR-----DQSPLVHYGWGDPIRLRHLYTSGPHGLSS 1.08 ± 0.33 ESLR-----DESPLVHYGWGDPIRLRHLYTSGPHGLSS 0.01 ± 0.01

Overall, more than 30 FGF19 variants were individually assessed fortheir tumorigenicity. A FGF19 variant carrying 3 amino acidsubstitutions (A30S, G31S, H33L) and a 5-amino acid deletion, referredas M70 (SEQ ID NO:1), was selected for further studies (FIG. 7A).

In contrast to FGF19, livers from db/db mice with high systemic exposureto M70 for 24 weeks were completely free of hepatic tumor nodules(15.6±2.8 tumor nodules per liver and 0.0∓0.0 tumor nodules per liverfor FGF19 and M70, respectively, n=5, p<0.001; FIG. 7B).FGF19-expressing mice exhibited significant increase in liver weight(2.91±0.19 g vs. 1.86±0.12 g in control mice, n=5, p<0.001; FIG. 7C),which as reported in previous studies closely correlates with livertumor burden. In contrast, M70-expressing mice did not show anyincreased liver weight (1.56±0.09 g vs. 2.91±0.19 g in FGF19 mice, n=5,p<0.001; FIG. 7C). Similar results were obtained when the liver-to-bodyweight ratio was calculated (FIG. 7D and FIG. 7E). Average serumconcentration of M70 was 2-3 μg/ml in these mice, about 10,000-foldhigher than circulating FGF19 level in human (FIG. 7F). Liverhistological analysis revealed that M70-expressing mice did not developany discernable preneoplastic and neoplastic lesions associated withFGF19 overexpression in mice. Specifically, no altered hepatic foci,hepatocellular dysplasia, hepatocellular adenomas, or hepatocellularcarcinomas was observed (FIG. 7G). In FGF19-expressing mice,non-tumorigenic regions showed increased cellular density around centralvein, but no such change was observed in M70-expressing mice.Overexpression of M70 did not cause increased number of Ki-67-positivecells resulting from FGF19-overexpression (FIG. 7G). Furthermore, whileliver tumor lesions in FGF19 expressing cells became highly positive forglutamine synthetase, no increased expression of glutamine synthease wasobserved in the liver of M70 expressing-mice (FIG. 7G). Finally, noliver toxicity was observed following 24 weeks of prolonged exposure toM70, as determined by serum levels of liver enzymes (FIG. 7H). Takentogether, these results demonstrate that M70 lacks the ability topromote hepatocellular tumorigenesis in db/db mice.

The tumorigenicity of M70 in a rasH2 transgenic mouse model was furtherevaluated. CB6F1-RasH2 mice hemizygous for a human H-RAS transgene havebeen extensively used as an accelerated evaluation for the conventional2-year carcinogenicity assessment in rodents (Storer et al., 2010,Toxicologic Pathol. 38, 51-61). Sensitive to both genotoxic andnongenotoxic carcinogens, rasH2 mice develop both spontaneous andinduced neoplasms earlier than wild type mice. This strain also providesa relevant genetic background for studying hepatocarcinogenicity sinceactivation of RAS signaling pathway is frequently observed in human HCC(Calvisi et al., 2006, Gastroenterol. 130, 1117-1128).

During the course of a 52-week study, rasH2 mice expressing FGF19 or M70had a significant reduction of body weight gain compared with controlmice (FIG. 8A). However, the morphology of the livers from FGF19 andM70-expressing groups showed dramatic differences. Gross morphologicalchanges with multiple tumor nodules were observed in the livers of miceexpressing FGF19, consistent with the formation of HCC (3.8+1.5 tumornodules per liver; FIG. 8B). In contrast, the livers from miceexpressing M70 had normal gross morphology and were completely free oftumor nodules (FIG. 8B). It should be pointed out that a low level ofspontaneous liver tumor formation was observed in control rasH2 mice(FIG. 8B). M70-expressing animals showed a dramatic decrease in liverweight compared with FGF19 mice (0.76+0.05 g vs. 1.71+0.24 g in FGF19mice, n=9, p<0.001; FIG. 8C). M70 also normalized the ratio of liver andbody weight in rasH2 mice (5.34+0.24% vs. 8.66+1.36% in FGF19 mice, n=9,p<0.01; FIG. 8D). The serum levels of FGF19 and M70 in these mice arecomparable, which are 155±28 ng/ml and 209±22 ng/ml, respectively (FIG.8E).

H & E stained liver sections from these mice were evaluated for thepresence of tumors and preneoplastic lesions (FIG. 8F). In addition,anti-glutamine synthetase staining was carried out as a marker ofFGF19-induced liver tumor (FIG. 8F). The sections stained for glutaminesynthetase were taken from paired section stained with H & E and thephotographs showed the same portal (p) and central (c) veins. rasH2 miceexpressing FGF19 displayed hepatocellular adenoma as well ashepatocellular carcinomas. Preneoplastic hepatocellular lesions werealso noted in FGF19-expressing rasH2 mice. Remarkably, none of thelivers from mice expressing M70 exhibited tumors or histologicalevidence of preneoplastic lesions (FIG. 8F). Corroborating histologicalresults, increased hepatic expression of Ki-67 and AFP (an embryonichepatic protein often induced in HCC (Marrero and El-Serag, 2011,Hepatol. 53, 1060-1062) were observed in FGF19-expressing rasH2 mice,but not in M70-expressing mice (FIG. 8G).

These results demonstrate that, unlike FGF19, prolonged exposure to highcirculating levels of M70 (i.e., 24 weeks in db/db mice or 52 weeks inrasH2 mice) does not promote liver tumor formation.

Example 9 M70 Binds and Activates FGFR4 In Vitro and In Vivo

To elucidate the molecular mechanism that underline M70's inability toinduce liver tumors, the interaction of M70 to the known receptorcomplex of FGF19 was assessed. Surface plasmon resonance (SPR) analysiswas used to measure direct binding of M70 or FGF19 to FGFR4. In aBiacore assay, M70 or FGF19 was used to flow over chips coated with anFc fusion protein of the extracellular domain (ECD) of FGFR4. M70directly interacted with FGFR4 with comparable affinity to FGF19(dissociation constant K_(D)=134±47 nM and 167±5 nM, respectively, FIG.9A and FIG. 9B). M70 also bound with similar affinity to KLB as FGF19(K_(D)=24.1+11.0 pM and 28.5+0.8 pM, respectively; data not shown). M70binds to the same site of KLB as FGF19, demonstrated by a competitionBiacore assay (data not shown). In a solid phase assay, M70 interactedwith FGFR4-KLB receptor complex (FIG. 9C). The presence of KLBdramatically increased ligand-receptor affinity. The dissociationconstant of M70 binding to the FGFR4-KLB receptor complex indicated ahigh-affinity interaction, with K_(D) of 2.14 nM (vs. K_(D) of 2.49 nMfor FGF19).

The ability of M70 to activate its receptors was evaluated in acell-based assay using rat L6 cells transfected with a FGF-responsiveGAL-Elk1 luciferase reporter gene (Wu et al., 2011, PloS one 6, e17868;Wu et al., 2010a, PNAS, 107, 14158-14163). In this assay, effectivebinding of a ligand to FGFR results in activation of an endogenous ERKkinase pathway, leading to subsequent activation of a chimerictranscriptional activator comprising of an Elk-1 activation domain and aGAL4 DNA-binding domain. L6 cells lack functional FGFR or KLB and areonly responsive to FGF19 when co-transfected with cognate receptors(data not shown). M70 activated intracellular signaling pathways in L6cells co-expressing FGFR4 and KLB as effectively as FGF19 (EC50=38 pMand 52 pM for M70 and FGF19, respectively; FIG. 9D). In contrast,signaling in cells transfected with FGFR4 alone was much less responsiveto either ligand, showing a >500-fold reduction in potency upon additionof either FGF19 or M70 (FIG. 9D). These results suggest that theformation of a ternary complex between FGFR4-KLB co-receptors and thecognate ligands is important for potent activation of intracellularsignaling. FGFR4 pathway activation in Hep3B, a human HCC cell line wasthen analyzed. Hep3B cells predominantly express FGFR4, among isoformsof FGFRs, and KLB. Recombinant M70 protein induced phosphorylation andactivation of ERK with a similar potency and efficacy as wild type FGF19(half maximum effective concentration EC₅₀=0.38 nM and 0.37 nM for M70and FGF19, respectively; FIG. 9E).

FGF19/FGF15 have been implicated in the regulation of hepatic bile acidmetabolism in humans and in rodents, respectively (Holt et al., 2003,Genes Dev. 17, 1581-1591) (Inagaki et al., 2005, Cell Metabol. 2,217-225). FGF19/FGF15 potently represses hepatic expression ofcholesterol-7a-hydroxylase 1 (Cyp7a1), in a process that requires FGFR4(Inagaki et al., 2005, Cell Metabol. 2, 217-225; Wu et al., 2011, PloSone 6, e17868). The ability of M70 to regulate Cyp7a1 in primaryhepatocytes was evaluated. Upon addition to the culture media, M70effectively repressed Cyp7a1 expression in primary hepatocytes derivedfrom mouse, rat, or human liver (FIG. 9F). The activity of M70 wascomparable to that of wild-type FGF19 (half maximum inhibitoryconcentration IC50 0.64 pM for M70 vs. 0.65 pM for FGF19 in primarymouse hepatocytes; IC50 0.49 pM for M70 vs. 3.96 pM for FGF19 in primaryrat hepatocytes; IC50 6.80 pM for M70 vs. 1.73 pM for FGF19 in primaryhuman hepatocytes; FIG. 9F). In primary human hepatocytes, the additionof FGF19 resulted in a maximum suppression of Cyp7a1 mRNA by 97%.Similarly, M70 was able to reduce Cyp7a1 expression by 98% (FIG. 9F).

To evaluate the acute effects of M70 administration on hepaticexpression of Cyp7a1 in vivo, mice were injected intraperitoneally(i.p.) with recombinant M70 or FGF19 protein at doses ranging from 0.001to 10 mg/kg (FIG. 9G). A single i.p. injection of M70 potentlysuppressed Cyp7a1 mRNA with an ED50 of 1.29 μg/kg (FIG. 9G). These datademonstrate that systemic administration of M70 can potently and rapidlytrigger FGFR4-mediated intracellular in vivo.

In summary, M70 and wild type FGF19 exhibit a comparable profile ofbiological activities, leading to activation of ERK signaling and Cyp7a1regulation.

Example 10 M70 Exhibits Differential Signaling Pathway ActivationCompared with FGF19

M70 binds FGFR4 receptor complex and activates the intracellularsignaling pathway leading to Cyp7a1 repression, but does not promoteliver tumor formation in either db/db or rasH2 mouse models. In order toelucidate the molecular basis for the lack of tumorigenic potential, theactivation of key signaling proteins involved in tumorigenesis,including ERK, PI3K/AKT, STATs, and WNT/β-catenin pathways, wasanalyzed.

M70 and FGF19 proteins (1 mg/kg) were injected intraperitoneally intodb/db mice. Livers were collected 15 minutes (data not shown), 2 hours(FIG. 10A), and 4 hours (data not shown) later and phosphorylation ofsignaling proteins was measured by immunoblotting. Consistent with theability of both molecules to signal in cultured primary hepatocytes,FGF19 and M70 stimulated ERK phosphorylation to a similar extent inliver tissues in vivo. In line with previous reports on the role ofFGF19 in modulating hepatic protein synthesis (Kir et al., 2011, Science331, 1621-1624), both wild type FGF19 and M70 induced robustphosphorylation of ribosomal S6 protein in the liver (FIG. 10 and datanot shown). This agrees with the notion that M70 retains activity onFGFR4-KLB receptor complex. Neither M70 nor FGF19 had any effect onhepatic levels of phosphorylated AKT. No activation of GSK3β andβ-catenin was observed at all three time points tested.

Remarkably, FGF19 induced STAT3 phosphorylation 2 hours after dosing(FIG. 10A). This effect lasted to 4 hours post dosing (data not shown).In contrast, M70 did not increase STAT3 phosphorylation (FIG. 10A).IL-6, a known STAT3 activator, was shown to be upregulated in FGF19-butnot M70-treated livers (FIG. 10B). The pSTAT3 activation by FGF19 islikely due to non-cell autonomous mechanisms on the liver, since noinduction of pSTAT3 was observed 15 minutes after protein injection orin primary mouse hepatocyte culture (data not shown). Corroborating withSTAT3 phosphorylation and activation, increased expression of STAT3target genes, including survivin, bcl-X_(L), and cyclin D1, was observedin rasH2 livers expressing FGF19, not M70 (FIG. 10C). Since STAT3 is anoncogene frequently activated in HCC (He and Karin, 2011, Cell Res. 21,159-168), its activation by FGF19 poses a plausible mechanism forFGF19-induced hepatocarcinogenicity. The inability of M70 to activateSTAT3 pathway could contribute to its lack of tumorigenicity in vivo.

Thus, M70 only activates a subset of signaling pathways downstream ofits receptors, a hallmark of selective modulators (Kenakin andChristopoulos, 2013, Nat. Rev. Drug Discov. 12, 205-21). Theidentification and characterization of M70 allow us to define twodistinct biological processes regulated by FGF19-FGFR4 pathway, bileacid homeostasis and tumorigenesis.

Example 11 M70 Inhibits FGF19-Mediated Tumor Formation

Our observation suggests that M70 behaves as a selective modulator or a“biased ligand” to activate the metabolic signaling but not thetumorigenic signals from FGFR4. Next, it was determined whether thebiased agonism of M70 can be utilized to inhibit FGF19-associated tumorformation via an orthosteric or allosteric mechanism.

db/db mice were injected with 3×10¹⁰ genome copies of AAV-FGF19, with orwithout 10-fold molar excess of AAV-M70 (3×10¹¹ genome copies). Micewere necropsied 24 weeks after transgene expression and the livers wereexercised for analysis. While ectopic expression of FGF19 in db/db micepromoted the formation of tumor nodules on hepatic surface (7.8+2.3tumor nodules per liver), livers from mice expressing both FGF19 and M70were completely free of tumor nodules (FIG. 11A). Liver weights fromM70-coexpressing mice were significantly lower relative toFGF19-expressing mice (1.59 g±0.14 g and 2.42 g±0.20 g, respectively,n=5, p<0.01; FIG. 11B). The ratios of liver to body weight in M70 andFGF19 co-treated mice were not significantly different from those ofcontrol mice (FIG. 11C). The serum levels of FGF19 were 94±12 ng/ml whendosed alone, and the combined serum level of FGF19 and M70 was 453±169ng/ml (FIG. 11D). Histological analysis of the livers confirmed thatunlike FGF19-expression mice, mice co-expressing M70 and FGF19 did notexhibit any histological evidence of liver tumors (FIG. 11E). These datademonstrate that M70 effectively competes with FGF19 to prevent tumorformation in FGF19-expressing mice.

FGF19 is reported to be amplified and/or overexpressed in HCC and coloncancer (Desnoyers et al., 2008; Sawey et al., 2011, Oncogene 27, 85-97).A panel of liver, colon, breast and other human cancer cell lines werescreened, and it was observed that FGF19 is produced and secreted by,among others, Huh-7 (HCC) and HCT-116 (colon cancer) cell lines (FIG.11F), which were chosen for further studies. The levels of FGF19 in theculture media reached 1-2 ng/ml by ELISA measurement, about 10-foldhigher than physiological FGF19 concentration in human.

HCC cell line Huh-7 harbors the 11q13.3 amplicon and overexpresses bothFGF19 and CCND1. The effect of M70 on the tumor-forming ability ofHuh-cells was tested. Athymic nude mice were injected subcutaneouslywith Huh-7 cells, and tumors were allowed to reach a size of ˜100 mm³.At that point, mice were placed into 2 treatment groups: one injectedintravenously with AAV-M70, another with a control virus. M70-treatedmice exhibited a trend of delayed growth by 28% (end-stage tumor size:1856±348 mm³ in controls vs. 1340±406 mm³ after M70 treatment; n=10;FIG. 11G). No significant effect on body weight was noted (data notshown).

The effect of M70 on HCT-116 colon cancer xenograft growth was alsoexaminer. Mice bearing established HCT116 colon cancer tumors were dosedwith AAV-M70 or control virus. As early as day 8 after treatment began,M70 suppressed tumor growth by 37% (tumor size: 459±83 mm³ in controlgroup vs. 287±87 mm³ in M70 group; n=5; FIG. 11H). On day 15 posttreatment, M70-treated mice exhibited a statistically significant 71%inhibition of tumor growth (end-stage tumor size: 1634±524 mm³ incontrols vs. 479±155 mm³ after M70 treatment, n=5, p<0.001; FIGS. 11Hand 11I). No significant effect on body weight was observed (FIG. 11J).

These results suggest that M70 acts as a biased ligand that is capableof antagonizing wild type FGF19 in tumorigenic signaling, anddemonstrate the potential of using a selective modulator such as M70 tosuppress FGF19-dependent tumor growth.

Example 12 M70 Inhibits CT26 Colon Tumor Growth

This study was conducted to further assess the effect of M70 on tumorprogression in a syngenic model in immune-competent mice. CT26 is amouse colon cancer cell line, which grafts and grows well in syngenicBalb/c mice. CT26 was widely used for characterizing compounds/agents ontumor growth, especially for evaluating cancer immunotherapies.

As a positive control, a blocking antibody against Programmed Death-1(PD-1) was used. PD-1 and its ligands PD-L1/PD-L2 represent an immunecheckpoint axis. The PD-1 pathway down-regulates tumor-specific immunityby impairing T-cell responses and promoting the induction of Foxp3+Tregs in the periphery. Blocking the PD-1 pathway, in conjunction withother immune therapies, inhibits tumor progression in syngenic models.Multiple human anti-PD-1 monoclonal antibodies (mAbs), as well as humananti-PD-L1 mAbs, have entered clinical trials, and the first anti-PD-1antibody was recently approved by FDA as an anti-cancer therapy.

Balb/c mice were purchased from the Jackson Laboratory. Animals weremaintained in a pathogen-free facility. All animal protocols wereapproved by Institutional Animal Care and Use Committee at NGMBiopharmaceuticals.

CT26 mouse colon cancer cell line was purchased from ATCC. Cells werecultured in DMEM with 10% FBS and penicillin/streptomycin cocktail.Exponentially grown cells were harvested for implantation in mice. Cellswere resuspended in saline for injection.

Balb/c mice were implanted with 1×10⁶ CT26 cells on the right flank.Three days later, M70 protein was subcutaneously injected in Balb/c micebearing the CT26 implant once daily for 15 days. The growth of CT26tumor was measured twice weekly with a caliper. Tumor volume wascalculated using formula: Tumor volume=width²*length/2.

As shown in FIG. 13, M70 delays tumor growth in a CT26 colon cancersyngenic mouse model following administration of 10 mg/kg doses (FIG.13A) or 3 mg/kg doses (FIG. 13B) as compared to vehicle alone. M70 wasalso shown to reduce body weight following administration of 10 mg/kgdoses (FIG. 14A) or 3 mg/kg doses (FIG. 14B).

Thus, these studies show that M70 treatment delays CT26 colon tumorgrowth in immune-competent Balb/c syngenic mice, with anti-tumorefficacy being observed for both doses (3 mg/kg and 10 mg/kg) of M70.

Particular embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Upon reading the foregoing description, variations of the disclosedembodiments may become apparent to individuals working in the art, andit is expected that those skilled artisans may employ such variations asappropriate. Accordingly, it is intended that the invention be practicedotherwise than as specifically described herein, and that the inventionincludes all modifications and equivalents of the subject matter recitedin the claims appended hereto as permitted by applicable law. Moreover,any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

All publications, patent applications, accession numbers, and otherreferences cited in this specification are herein incorporated byreference in its entirety as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention. Further, the dates of publication providedcan be different from the actual publication dates which can need to beindependently confirmed.

SEQUENCE LISTING

The present specification is being filed with a computer readable form(CRF) copy of the Sequence Listing in ASCII text format submitted viaEFS-Web. The CRF copy of the Sequence Listing, entitled13370-092-999_Sequence_Listing.txt, which was created on Jun. 11, 2019and is 50,262 bytes in size, is incorporated herein by reference in itsentirety.

1.-72. (canceled)
 73. A method for determining whether a test subjecthaving a metabolic disorder is a candidate for treatment with a FGF19variant, comprising: (i) (a) co-administering FGF19 or a FGF19surrogate, and a FGF19 variant to the test subject having a metabolicdisorder, wherein the amount of the FGF19 or the FGF19 surrogateadministered to the test subject is sufficient to induce a cancerouscondition in a reference population, and (b) determining whether anindicia of a cancerous condition is observed; wherein the absence of theindicia indicates that the test subject is a candidate for treatmentwith a FGF19 variant; (ii) (a) providing a test subject having anindicia of a cancerous condition, the subject having a metabolicdisorder, co-administering FGF19 or a FGF19 surrogate, and a FGF19variant to the test subject, wherein the amount of the FGF19 or theFGF19 surrogate administered to the test subject is sufficient to inducea cancerous condition in a reference population, and (b) determiningwhether the indicia of a cancerous condition is enhanced in the testsubject; wherein the absence of enhancement of the indicia of acancerous condition indicates that the test subject is a candidate fortreatment with a FGF19 variant; or (iii) (a) providing a test subjecthaving an indicia of a cancerous condition, the test subject having ametabolic disorder, co-administering FGF19 or a FGF19 surrogate, and aFGF19 variant to the test subject, wherein the amount of the FGF19 orthe FGF19 surrogate is administered to the test subject is sufficient toinduce a cancerous condition in a reference population, and (b)determining whether the indicia of a cancerous condition is reduced inthe test subject; wherein the reduction of the indicia of a cancerouscondition indicates that the test subject is a candidate for treatmentwith a FGF19 variant.
 74. The method of claim 73, wherein the FGF19variant comprises or consists of an amino acid sequence set forth in anyone of SEQ ID NOs:1 and 5-29; or a subsequence or fragment thereof. 75.The method of claim 73, wherein the indicia of a cancerous condition isa tumor.
 76. The method of claim 73, wherein the test subject has anincreased level of mature FGF19 compared to the level of mature FGF19 ina sample population.
 77. The method of claim 7373, wherein the FGF19variant polypeptide improves at least one condition selected from thegroup consisting of a hyperglycemic condition, insulin resistance,hyperinsulinemia, glucose intolerance, metabolic syndrome, obesity, andan undesirable body mass.
 78. The method of claim 73, wherein at leastone of the FGF19 or the FGF19 surrogate, and the FGF19 variant islabeled.
 79. The method of claim 7373, wherein FGF19 is co-administeredwith the FGF19 variant.
 80. The method of claim 73, wherein thedetermining step is performed more than 20 weeks, more than 9 months ormore than 12 months after the co-administering step.
 81. A method oftreating a subject having a metabolic disorder, comprising: (a)providing a subject having a metabolic disorder, wherein the subjectexhibits an indicia of a FGF19-induced cancerous condition, and (b)administering to the subject a therapeutically effective amount of aFGF19 variant, wherein the FGF19 variant comprises or consists of anamino acid sequence set forth in any one of SEQ ID NOs:1 and 5-29; or asubsequence or fragment thereof; wherein there is an improvement in themetabolic disorder in the subject.
 82. The method of claim 81, wherein(i) the subject is an animal; (ii) the cancerous condition is a tumor;(iii) the metabolic disorder is selected from the group consisting of ahyperglycemic condition, insulin resistance, hyperinsulinemia, glucoseintolerance, obesity and metabolic syndrome; or (iv) the improvement inthe metabolic disorder in the subject is a decrease in blood glucose, adecrease in body weight or a decrease in insulin.
 83. A method ofpreventing a FGF19-dependent cancer or tumor, or a symptom thereof, in asubject, comprising administering to the subject a therapeuticallyeffective amount of a FGF19 variant, thereby preventing theFGF19-dependent cancer or tumor, or symptom thereof in the subject. 84.The method of claim 83, wherein the FGF19-dependent cancer or tumor ishepatocellular carcinoma, a colon cancer or tumor, a prostate cancer ortumor, or a lung cancer or tumor.
 85. The method of claim 83, whereinthe FGF19 variant is a polypeptide comprising or consisting of an aminoacid sequence set forth in SEQ ID NO:1.
 86. The method of claim 83,wherein the subject is a subject in need thereof.